Search

Mucus and its movements are essential to epithelial tissue immune defenses against pathogens, including fungal pathogens, which can infect respiratory, gastrointestinal or the genito-urinary tracts. Several epithelial cell types contribute to their immune defense. This review focuses on the respiratory tract because of its paramount importance, but the observations will apply to epithelial cell defenses of other mucosal tissue, including the gastrointestinal and genito-urinary tracts. Mucus and its movements can enhance or degrade the immune defenses of the respiratory tract, particularly the lungs. The enhancements include inhaled pathogen entrapments, including fungal pathogens, pollutants and particulates, for their removal. The detriments include smaller lung airway obstructions by mucus, impairing the physical removal of pathogens and impairing vital transfers of oxygen and carbon dioxide between the alveolar circulatory system and the pulmonary air. Inflammation, edema and/or alveolar cellular damage can also reduce vital transfers of oxygen and carbon dioxide between the lung alveolar circulatory system and the pulmonary air. Furthermore, respiratory tract defenses are affected by several fatty acid mediators which activate cellular receptors to manipulate neutrophils, macrophages, dendritic cells, various innate lymphoid cells including the natural killer cells, T cells, γδ T cells, mucosal-associated invariant T cells, NKT cells and mast cells. These mediators include the inflammatory and frequently immunosuppressive prostaglandins and leukotrienes, and the special pro-resolving mediators, which normally resolve inflammation and immunosuppression. The total effects on the various epithelial cell and immune cell types, after exposures to pathogens, pollutants or particulates, will determine respiratory tract health or disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tuberculosis caused by the pathogen Mycobacterium tuberculosis leads to increased mortality and morbidity worldwide. The prevalence of highly drug-resistant strains has reinforced the need for greater understanding of host-pathogen interactions at the cellular and molecular levels. Our previous work demonstrated critical roles of calcium ion channels in regulating protective responses to mycobacteria. In this report, we deciphered the roles of inwardly rectifying K+ ion channel Kir2.1 in epithelial cells. Data showed that infection of epithelial cells (and macrophages) increases the surface expression of Kir2.1. This increased expression of Kir2.1 results in higher intracellular mycobacterial survival, as either inhibiting or knocking down Kir2.1 results in mounting of a higher oxidative burst leading to a significant attenuation of mycobacterial survival. Further, inhibiting Kir2.1 also led to increased expression of T cell costimulatory molecules accompanied with increased activation of MAP kinases and transcription factors nuclear factor κB and phosphorylated CREB. Furthermore, inhibiting Kir2.1 induced increased autophagy and apoptosis that could also contribute to decreased bacterial survival. Interestingly, an increased association of heat shock protein 70 kDa with Kir2.1 was observed. These results showed that mycobacteria modulate the expression and function of Kir2.1 in epithelial cells to its advantage., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Mycobacterium bovis (M. bovis) Bacillus Calmette-Guerin (BCG) strain used in immunotherapy of bladder cancer (onco-BCG) due to its acid tolerance can be a candidate for prevention or reversion of deleterious effects towards gastric cell barrier initiated by gastric pathogen Helicobacter pylori (Hp) with high resistance to commonly used antibiotics. Colonization of gastric mucosa by Hp promotes oxidative stress, apoptosis resulting in the gastric barrier damage. The aim of this study was to examine the ability of onco-BCG bacilli to control the Hp driven gastric damage using the model of Cavia porcellus primary gastric epithelial cells or fibroblasts in vitro. These cells were treated with Hp surface antigens (glycine acid extract-GE or lipopolysaccharide-LPS) alone or with onco-BCG bacilli and evaluated for cell apoptosis and proliferation in conjunction with the level of soluble lipid peroxidation marker (s4HNE). The cell migration was determined by "wound healing assay", while cytokine response of cells, including interleukin (IL)-33, IL-1β, IL-8 and tumor necrosis factor alpha (TNF-α), by the ELISA. The apoptosis of cells pulsed in vitro with Hp surface components present in GE or with LPS was reduced after exposure of cells to mycobacteria. Similarly, the cell regeneration which was diminished by Hp LPS has been improved in response to mycobacteria. This study reveals that vaccine mycobacteria may reduce gastric barrier damage induced by Hp infection., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes., (© 2024 Wiley Periodicals LLC.)

Outer membrane vesicles (OMVs) are nanometer-sized membrane blebs secreted by all Gram-negative bacteria to facilitate bacterial communication and modulate the external environment, including in the context of host-microbe interactions. Neisseria gonorrhoeae releases OMVs during interactions with epithelial cells; however, beneficial functional activities for these OMVs have not yet been demonstrated. Our recent study shows that gonococcal OMVs are endocytosed by epithelial cells and subsequently induce mitophagy through a dual PorB-dependent mechanism. PorB is the major gonococcal outer membrane porin protein, which is able to translocate to mitochondria and dissipate the mitochondrial membrane potential, leading to the initiation of a conventional mitophagy mechanism that is dependent on PINK1 and the receptor proteins OPTN or CALCOCO2/NDP52. A second SQSTM1/p62-dependent mitophagy pathway results from direct K63-linked polyubiquitination of PorB lysine residue 171 by the E3 ubiquitin ligase RNF213. Induction of mitophagy favors intracellular gonococcal survival, because it reduces the release of bactericidal mitochondrial reactive oxygen species. These findings highlight a sophisticated bimodal PorB-dependent mechanism by which gonococcal OMVs modulate the intracellular environment to enhance survival in this hostile niche.

Interaction between viruses and bacteria during the development of infectious diseases is a complex question that requires continuous study. In this study, we explored the interactions between pseudorabies virus (PRV) and Pasteurella multocida (PM), which are recognized as the primary and secondary agents of porcine respiratory disease complex (PRDC), respectively. In vivo tests using mouse models demonstrated that intranasal inoculation with PRV at a sublethal dose induced disruption of murine respiratory barrier and promoted the invasion and damages caused by PM through respiratory infection. Inoculation with PRV also disrupted the barrier function of murine and porcine respiratory epithelial cells, and accelerated the adherence and invasion of PM to the cells. In mechanism, PRV infection resulted in decreased expression of tight junction proteins (ZO-1, occludin) and adherens junction proteins (β-catenin, E-cadherin) between neighboring respiratory epithelial cells. Additionally, PRV inoculation at an early stage downregulated multiple biological processes contributing to epithelial adhesion and barrier functions while upregulating signals beneficial for respiratory barrier disruption (e.g., the HIF-1α signaling). Furthermore, PRV infection also stimulated the upregulation of cellular receptors (CAM5, ICAM2, ACAN, and DSCAM) that promote bacterial adherence. The data presented in this study provide insights into the understanding of virus-bacteria interactions in PRDC and may also contribute to understanding the mechanisms of secondary infections caused by different respiratory viruses (e.g., influenza virus and SARS-CoV-2) in both medical and veterinary medicine., Importance: Co-infections caused by viral and bacterial agents are common in both medical and veterinary medicine, but the related mechanisms are not fully understood. This study investigated the interactions between the zoonotic pathogens PRV and PM during the development of respiratory infections in both cell and mouse models, and reported the possible mechanisms which included: (i) the primary infection of PRV may induce the disruption and/or damage of mammal respiratory barrier, thereby contributing to the invasion of PM; (ii) PRV infection at early stage accelerates the transcription and/or expression of several cellular receptors that are beneficial for bacterial adherence. This study may shed a light on understanding the mechanisms on the secondary infection of PM promoted by different respiratory viruses (e.g., influenza virus and SARS-CoV-2) in both medical and veterinary medicine., Competing Interests: The authors declare no conflict of interest.

Several reports suggest that intestinal tissue may be a natural niche for Chlamydia trachomatis infection and a reservoir for persistent infections in the human body. Due to the human specificity of the pathogen and the lack of suitable host models, there is limited knowledge on this topic. In our study, we modelled the course of the chlamydial infection in human primary gastrointestinal (GI) epithelial cells originating from patient-derived organoids. We show that GI cells are resistant to apical infection and C. trachomatis needs access to the basolateral membrane to establish an infection. Transmission electron microscopy analysis reveals the presence of both normal as well as aberrant chlamydial developmental forms in the infected cells, suggesting a possible cell-type specific nature of the infection. Furthermore, we show that the plasmid-encoded Pgp3 is an important virulence factor for the infection of human GI cells. This is the first report of C. trachomatis infection in human primary intestinal epithelial cells supporting a possible niche for chlamydial infection in the human intestinal tissue., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hovhannisyan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In the current study, two Salmonella Typhimurium strains, JOL 912 and JOL 1800, were engineered from the wild-type JOL 401 strain through in-frame deletions of the lon and cpxR genes, with JOL 1800 also lacking rfaL . These deletions significantly attenuated the strains, impairing their intracellular survival and creating unique immunological profiles. This study investigates the response of these strains to various abiotic stress conditions commonly experienced in vivo, including temperature, acidity, osmotic, and oxidative stress. Notably, cold stress induced a non-significant trend towards increased invasion by Salmonella compared to other stressors. Despite the observed attenuation, no significant alterations in entry mechanisms (trigger vs. zipper) were noted between these strains, although variations were evident depending on the host cell type. Both strains effectively localized within the cytoplasm, demonstrating their ability to invade and interact with the intracellular environment. Immunologically, JOL 912 elicited a robust response, marked by substantial activation of nuclear factor kappa B (NF-kB), and chemokines, interleukin 8 (CXCL 8) and interleukin 10 (CXCL 10), comparable to the wild-type JOL 401 (over a fourfold increase compared to JOL 1800). In contrast, JOL 1800 exhibited a minimal immune response. Additionally, these attenuations influenced the expression of cyclins D1 and B1 and caspases 3 and 7, indicating cell cycle arrest at the G2/M phase and promotion of the G0/G1 to S phase transition, alongside apoptosis in infected cells. These findings provide valuable insights into the mechanisms governing the association, internalization, and survival of Salmonella mutants, enhancing our understanding of their regulatory effects on host cell physiology.

Candida albicans, an opportunistic oral pathogen, synergizes with Staphylococcus aureus , allowing bacteria to co-invade and systemically disseminate within the host. Studying human-microbe interactions creates the need for a universal culture medium that supports fungal, bacterial, and human cell culturing, while allowing sensitive analytical approaches such as OMICs and chromatography techniques. In this study, we established a fully defined, customizable adaptation of Dulbecco's modified Eagle medium (DMEM), allowing multi-kingdom culturing of S. aureus , C. albicans , and human oral cell lines, whereas minimal version of DMEM (mDMEM) did not support growth of S. aureus , and neither did supplementation with dextrose, MEM non-essential amino acids, pyruvate, and Glutamax. This new medium composition, designated as "mDMEM-DMP," promoted growth of all tested S. aureus strains. Addition of 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) further improved growth, while higher concentrations did not improve growth any further. Higher concentrations of HEPES did result in prolonged stabilization of medium pH. mDMEM-DMP promoted (hyphal) C. albicans monoculturing and co-culturing on both solid and semi-solid surfaces. In contrast to S. aureus , addition of HEPES reduced C. albicans maximum culture optical density (OD). Finally, only buffered mDMEM-DMP (100 mM HEPES) was successful in maintaining the metabolic activity of human oral Ca9-22 and HO1N1 cell lines for 24 hours. Altogether, our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions., Importance: Interaction between microbes and the host are in the center of interest both in disease and in health. In order to study the interactions between microbes of different kingdoms and the host, alternative media are required. Synthetic media are useful as they allow addition of specific components. In addition, well-defined media are required if high-resolution analyses such as metabolomics and proteomics are desired. We describe the development of a synthetic medium to study the interactions between C. albicans, S. aureus, and human oral epithelial cells. Our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions., Competing Interests: The authors declare no conflict of interest.

Background: Group B Streptococcus (GBS) is the leading cause of neonatal meningitis responsible for a substantial cause of death and disability worldwide. The vast majority of GBS neonatal meningitis cases are due to the CC17 hypervirulent clone. However, the cellular and molecular pathways involved in brain invasion by GBS CC17 isolates remain largely elusive. Here, we studied the specific interaction of the CC17 clone with the choroid plexus, the main component of the blood-cerebrospinal fluid (CSF) barrier., Methods: The interaction of GBS CC17 or non-CC17 strains with choroid plexus cells was studied using an in vivo mouse model of meningitis and in vitro models of primary and transformed rodent choroid plexus epithelial cells (CPEC and Z310). In vivo interaction of GBS with the choroid plexus was assessed by microscopy. Bacterial invasion and cell barrier penetration were examined in vitro, as well as chemokines and cytokines in response to infection., Results: GBS CC17 was found associated with the choroid plexus of the lateral, 3rd and 4th ventricles. Infection of choroid plexus epithelial cells revealed an efficient internalization of the bacteria into the cells with GBS CC17 displaying a greater ability to invade these cells than a non-CC17 strain. Internalization of the GBS CC17 strain involved the CC17-specific HvgA adhesin and occurred via a clathrin-dependent mechanism leading to transcellular transcytosis across the choroid plexus epithelial monolayer. CPEC infection resulted in the secretion of several chemokines, including CCL2, CCL3, CCL20, CX3CL1, and the matrix metalloproteinase MMP3, as well as immune cell infiltration., Conclusion: Our findings reveal a GBS strain-specific ability to infect the blood-CSF barrier, which appears to be an important site of bacterial entry and an active site of immune cell trafficking in response to infection., (© 2024. The Author(s).)

The collective involvement of virulence markers of Escherichia coli as an emerging pathogen associated with periodontitis remains unexplained. This study aimed to implement an in vitro model of infection using a human epithelial cell line to determine the virulome expression related to the antibiotic and disinfectant resistance genotype and pulse field gel electrophoresis (PFGE) type in E. coli strains isolated from patients with periodontal diseases. We studied 100 strains of E. coli isolated from patients with gingivitis (n = 12), moderate periodontitis (n = 59), and chronic periodontitis (n = 29). The identification of E. coli and antibiotic and disinfectant resistance genes was performed through PCR. To promote the expression of virulence genes in the strains, an in vitro infection model was used in the human epithelial cell line A549. RNA was extracted using the QIAcube robotic equipment and reverse transcription to cDNA was performed using the QuantiTect reverse transcription kit (Qiagen). The determination of virulence gene expression was performed through real-time PCR. Overall, the most frequently expressed adhesion genes among the isolated strains of gingivitis, moderate periodontitis, and chronic periodontitis were fimH (48%), iha (37%), and papA (18%); those for toxins were usp (33%); those for iron acquisition were feoB (84%), fyuA (62%), irp-2 (61%), and iroN (35%); those for protectins were traT (50%), KpsMT (35%), and ompT (28%); and those for pathogenicity islands were malX (45%). The most common antibiotic and disinfectant resistance genes among gingivitis, moderate periodontitis, and chronic periodontitis strains were sul-2 (43%), bla SHV (47%), blaTEM (45%), tet(A) (41%), dfrA1 (32%), marR-marO (57%), and qacEA1 (79%). The findings revealed the existence of a wide distribution of virulome expression profiles related to the antibiotic and disinfectant resistance genotype and PFGE type in periodontal strains of E. coli. These findings may contribute toward improving the prevention and treatment measures for periodontal diseases associated with E. coli., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Monroy-Pérez, Hernández-Jaimes, Morales-Espinosa, Delgado, Martínez-Gregorio, García-Cortés, Herrera-Gabriel, De Lira-Silva, Vaca-Paniagua and Paniagua-Contreras.)

Background: Helicobacter pylori (H. pylori) infection is critical in the development and occurrence of gastric cancer. H. pylori secretes gamma-glutamyl transferase (GGT), which affects energy metabolism and histone methylation in mesenchymal stem cells. However, its effect on human gastric epithelial cells remains unclear. This study aimed to investigate the effects of GGT on energy metabolism and histone methylation in gastric epithelial cells and determine its role in the development and progression of H. pylori-induced gastric cancer., Methods: A GGT knockout H. pylori strain and mouse gastric cancer model were constructed, and alpha-ketoglutarate (α-KG) was added. The underlying mechanism was investigated using proteomics, immunohistochemistry, Western blotting, and other experimental assays., Results: H. pylori can colonize the host's stomach and destroy the gastric epithelium. GGT secreted by H. pylori decreased the concentration of glutamine in the stomach and increased H3K9me3 and H3K27me3 expression, which promoted the proliferation and migration of gastric epithelial cells. Additionally, α-KG reversed this effect. GGT increased the tumorigenic ability of nude mice. GGT, secreted by H. pylori, promoted the expression of ribosomal protein L15 (RPL15), while GGT knockout and supplementation with α-KG and trimethylation inhibitors reduced RPL15 expression and Wnt signaling pathway expression., Conclusions: H. pylori secreted GGT decreased the expression of glutamine and α-KG in gastric epithelial cells, increased the expression of histones H3K9me3 and H3K27me3, and activated the Wnt signaling pathway through RPL15 expression, ultimately changing the biological characteristics of the gastric epithelium and promoting the occurrence of gastric cancer. Altered energy metabolism and histone hypermethylation are important factors involved in this process., (© 2024. The Author(s).)

Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the K D value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection.IMPORTANCEBacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death., Competing Interests: V.V., S.U.L., W.P., E.B., K.S., V.M., N.N., and D.M. are employees of GSK. R.R., V.M., K.S., and D.M. report ownership of GSK shares and/or restricted GSK shares. M.M. is an employee of the University of Naples Federico II with a consultancy contract with GSK.

The egress of intracellular bacteria from host cells and cellular tissues is a critical process during the infection cycle. This process is essential for bacteria to spread inside the host and can influence the outcome of an infection. For the obligate intracellular Gram-negative zoonotic bacterium Chlamydia psittaci, little is known about the mechanisms resulting in bacterial egress from the infected epithelium. Here, we describe and characterize Chlamydia -containing spheres (CCSs), a novel and predominant type of non-lytic egress utilized by Chlamydia spp. CCSs are spherical, low-phase contrast structures surrounded by a phosphatidylserine-exposing membrane with specific barrier functions. They contain infectious progeny and morphologically impaired cellular organelles. CCS formation is a sequential process starting with the proteolytic cleavage of a DEVD tetrapeptide-containing substrate that can be detected inside the chlamydial inclusions, followed by an increase in the intracellular calcium concentration of the infected cell. Subsequently, blebbing of the plasma membrane begins, the inclusion membrane destabilizes, and the proteolytic cleavage of a DEVD-containing substrate increases rapidly within the whole infected cell. Finally, infected, blebbing cells detach and leave the monolayer, thereby forming CCS. This sequence of events is unique for chlamydial CCS formation and fundamentally different from previously described Chlamydia egress pathways. Thus, CCS formation represents a major, previously uncharacterized egress pathway for intracellular pathogens that could be linked to Chlamydia biology in general and might influence the infection outcome in vivo .IMPORTANCEHost cell egress is essential for intracellular pathogens to spread within an organism and for host-to-host transmission. Here, we characterize Chlamydia -containing sphere (CCS) formation as a novel and predominant non-lytic egress pathway of the intracellular pathogens Chlamydia psittaci and Chlamydia trachomatis . CCS formation is fundamentally different from extrusion formation, the previously described non-lytic egress pathway of C. trachomatis . CCS formation is a unique sequential process, including proteolytic activity, followed by an increase in intracellular calcium concentration, inclusion membrane destabilization, plasma membrane blebbing, and the final detachment of a whole phosphatidylserine-exposing former host cell. Thus, CCS formation represents an important and previously uncharacterized egress pathway for intracellular pathogens that could possibly be linked to Chlamydia biology, including host tropism, protection from host cell defense mechanisms, or bacterial pathogenicity., Competing Interests: The authors declare no conflict of interest.

Candida albicans causes millions of mucosal infections in humans annually. Hyphal overgrowth on mucosal surfaces is frequently associated with tissue damage caused by candidalysin, a secreted peptide toxin that destabilizes the plasma membrane of host cells thereby promoting disease and immunopathology. Candidalysin was first identified in C. albicans strain SC5314, but recent investigations have revealed candidalysin "variants" of differing amino acid sequence in isolates of C. albicans , and the related species C. dubliniensis , and C tropicalis , suggesting that sequence variation among candidalysins may be widespread in natural populations of these Candida species. Here, we analyzed ECE1 gene sequences from 182 C . albicans isolates, 10 C . dubliniensis isolates, and 78 C . tropicalis isolates and identified 10, 3, and 2 candidalysin variants in these species, respectively. Application of candidalysin variants to epithelial cells revealed differences in the ability to cause cellular damage, changes in metabolic activity, calcium influx, MAPK signalling, and cytokine secretion, while biophysical analyses indicated that variants exhibited differences in their ability to interact with and permeabilize a membrane. This study identifies candidalysin variants with differences in biological activity that are present in medically relevant Candida species., Importance: Fungal infections are a significant burden to health. Candidalysin is a toxin produced by Candida albicans that damages host tissues, facilitating infection. Previously, we demonstrated that candidalysins exist in the related species C. dubliniensis and C. tropicalis , thereby identifying these molecules as a toxin family. Recent genomic analyses have highlighted the presence of a small number of candidalysin "variant" toxins, which have different amino acid sequences to those originally identified. Here, we screened genome sequences of isolates of C. albicans , C. dubliniensis , and C. tropicalis and identified candidalysin variants in all three species. When applied to epithelial cells, candidalysin variants differed in their ability to cause damage, activate intracellular signaling pathways, and induce innate immune responses, while biophysical analysis revealed differences in the ability of candidalysin variants to interact with lipid bilayers. These findings suggest that intraspecies variation in candidalysin amino acid sequence may influence fungal pathogenicity., Competing Interests: The authors declare no conflict of interest.

The conjunctiva has immune-responsive properties to protect the eye from infections. Its innate immune system reacts against external pathogens, such as fungi. The complement factor C5a is an important contributor to the initial immune response. It is known that activation of transient-receptor-potential-vanilloid 1 (TRPV1) and TRP-melastatin 8 (TRPM8) channels is involved in different immune reactions and inflammation in the human body. The aim of this study was to determine if C5a and mucor racemosus e voluminae cellulae (MR) modulate Ca 2+ -signaling through changes in TRPs activity in human conjunctival epithelial cells (HCjECs). Furthermore, crosstalk was examined between C5a and MR in mediating calcium regulation. Intracellular Ca 2+ -concentration ([Ca 2+ ] i ) was measured by fluorescence calcium imaging, and whole-cell currents were recorded using the planar-patch-clamp technique. MR was used as a purified extract. Application of C5a (0.05-50 ng/mL) increased both [Ca 2+ ] i and whole-cell currents, which were suppressed by either the TRPV1-blocker AMG 9810 or the TRPM8-blocker AMTB (both 20 µM). The N-terminal peptide C5L2p (20-50 ng/mL) blocked rises in [Ca 2+ ] i induced by C5a. Moreover, the MR-induced rise in Ca 2+ -influx was suppressed by AMG 9810 and AMTB, as well as 0.05 ng/mL C5a. In conclusion, crosstalk between C5a and MR controls human conjunctival cell function through modulating interactions between TRPV1 and TRPM8 channel activity.

More than two million people worldwide are affected by life-threatening, invasive fungal infections annually. Candida species are the most common cause of nosocomial, invasive fungal infections and are associated with mortality rates above 40%. Despite the increasing incidence of drug-resistance, the development of novel antifungal formulations has been limited. Here we investigate the antifungal mode of action and therapeutic potential of positively charged, synthetic peptide mimics to combat Candida albicans infections. Our data indicates that these synthetic polymers cause endoplasmic reticulum stress and affect protein glycosylation, a mode of action distinct from currently approved antifungal drugs. The most promising polymer composition damaged the mannan layer of the cell wall, with additional membrane-disrupting activity. The synergistic combination of the polymer with caspofungin prevented infection of human epithelial cells in vitro, improved fungal clearance by human macrophages, and significantly increased host survival in a Galleria mellonella model of systemic candidiasis. Additionally, prolonged exposure of C. albicans to the synergistic combination of polymer and caspofungin did not lead to the evolution of tolerant strains in vitro. Together, this work highlights the enormous potential of these synthetic peptide mimics to be used as novel antifungal formulations as well as adjunctive antifungal therapy., (© 2024. The Author(s).)

Like TNFα, IL-6 is upregulated in Crohn's disease (CD) especially in patients associated with Mycobacterium avium paratuberculosis (MAP) infection, and both cytokines have been targeted as a therapeutic option for the treatment of the disease despite the accepted partial response in some patients. Limited response to anti-IL-6 receptor-neutralizing antibodies therapy may be related to the homeostatic dual role of IL-6. In this study, we investigated the effects and the signaling mechanism of IL-6 involved in intestinal epithelial integrity and function during MAP infection using an in vitro model that consists of THP-1, HT-29 and Caco-2 cell lines. Clinically, we determined that plasma samples from MAP-infected CD patients have higher IL-6 levels compared to controls (P-value < 0.001). In CD-like macrophages, MAP infection has significantly upregulated the secretion of IL-6 and the shedding of (IL-6R) from THP-1 macrophages, P-value < 0.05. Intestinal cell lines (Caco-2 and HT-29) were treated with the supernatant of MAP-infected THP-1 macrophages with or without a neutralizing anti-IL-6R antibody. Treating intestinal Caco-2 cells with supernatant of MAP-infected macrophages resulted in significant upregulation of intestinal damage markers including claudin-2 and SERPINE1/PAI-1. Interestingly, blocking IL-6 signaling exacerbated that damage and further increased the levels of the damage markers. In HT-29 cells, MAP infection upregulated MUC2 expression, a protective response that was reversed when IL-6R was neutralized. More importantly, blocking IL-6 signaling during MAP infection rescued damaged Caco-2 cells from MAP-induced apoptosis. The data clearly supports a protective role of IL-6 in intestinal epithelia integrity and function especially in CD patients associated with MAP infection. The findings may explain the ineffective response to anti-IL6 based therapy and strongly support a therapeutic option that restores the physiologic level of IL-6 in patient's plasma. A new treatment strategy based on attenuation of IL-6 expression and secretion in inflammatory diseases should be considered., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Alhendi and Naser.)

Helicobacter pylori is a microaerophilic Gram-negative bacterium that resides in the human stomach and is classified as a class I carcinogen for gastric cancer. Numerous studies have demonstrated that H. pylori infection plays a role in regulating the function of host cells, thereby contributing to the malignant transformation of these cells. However, H. pylori infection is a chronic process, and short-term cellular experiments may not provide a comprehensive understanding of the in vivo situation, especially when considering the lower oxygen levels in the human stomach. In this study, we aimed to investigate the mechanisms underlying gastric cell dysfunction after prolonged exposure to H. pylori under hypoxic conditions. We conducted a co-culture experiment using the gastric cell line GES-1 and H. pylori for 30 generations under intermittent hypoxic conditions. By closely monitoring cell proliferation, migration, invasion, autophagy, and apoptosis, we revealed that sustained H. pylori stimulation under hypoxic conditions significantly influences the function of GES-1 cells. This stimulation induces epithelial-mesenchymal transition and contributes to the propensity for malignant transformation of gastric cells. To confirm the in vitro results, we conducted an experiment involving Mongolian gerbils infected with H. pylori for 85 weeks. All the results strongly suggest that the Nod1 receptor signaling pathway plays a crucial role in H. pylori -related apoptosis and autophagy. In summary, continuous stimulation by H. pylori affects the functioning of gastric cells through the Nod1 receptor signaling pathway, increasing the likelihood of cell carcinogenesis. The presence of hypoxic conditions further exacerbates this process.IMPORTANCEDeciphering the collaborative effects of Helicobacter pylori infection on gastric epithelial cell function is key to unraveling the development mechanisms of gastric cancer. Prior research has solely examined the outcomes of short-term H. pylori stimulation on gastric epithelial cells under aerobic conditions, neglecting the bacterium's nature as a microaerophilic organism that leads to cancer following prolonged stomach colonization. This study mimics a more genuine in vivo infection scenario by repeatedly exposing gastric epithelial cells to H. pylori under hypoxic conditions for up to 30 generations. The results show that chronic exposure to H. pylori in hypoxia substantially increases cell migration, invasion, and epithelial-mesenchymal transition, while suppressing autophagy and apoptosis. This highlights the significance of hypoxic conditions in intensifying the carcinogenic impact of H. pylori infection. By accurately replicating the in vivo gastric environment, this study enhances our comprehension of H. pylori 's pathogenic mechanisms in gastric cancer., Competing Interests: The authors declare no conflict of interest.

Aims: To evaluate the prevalence, molecular characteristics, antimicrobial susceptibility, and epithelial invasion of Streptococcus agalactiae strains isolated from pregnant women and newborns in Rio de Janeiro, Brazil., Methods and Results: A total of 67 S. agalactiae isolates, 48 isolates from pregnant women and 19 from neonates, were analyzed. Capsular type Ia and V were predominant (35.8%/each). The multilocus sequence typing analysis revealed the presence of 19 STs grouped into 6 clonal complexes with prevalence of CC17/40.3% and CC23/34.3%. The lmb and iag virulence genes were found in 100% of isolates. Four S. agalactiae strains, belonging to CC17/ST1249 and CC23/ST23, were able to adhere to A549 respiratory epithelial cells. Antimicrobial resistance was verified mainly to tetracycline (85%), erythromycin (70.8%), and clindamycin (58.3%). Four S. agalactiae isolates were multidrug resistant. The resistance genes tested were found in 92.5% of isolates for tetM, 58.2% for ermB, 28.4% for mefAE, and 10.4% for tetO., Conclusion: The study showed a high prevalence of virulence and antimicrobial genes in S. agalactiae strains isolated from pregnant women and newborns, supporting the idea that continued surveillance is necessary to identify risk factors and perform long-term follow-up in pregnant women and neonates in Rio de Janeiro., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Epithelial cells orchestrate immune responses against fungal pathogens. This review highlights advances in integrating epithelial cells in immune responses against inhaled molds and dimorphic fungi, and against Candida species that colonize mucosal surfaces. In the lung, epithelial cells respond to interleukin-1 (IL-1) and interferon signaling to regulate effector cell influx and fungal killing. In the alimentary and vulvovaginal tracts, epithelial cells modulate fungal commensalism, invasive growth, and local immune tone, in part by responding to damage caused by candidalysin, a C. albicans peptide toxin, and through IL-17-dependent release of antimicrobial peptides that contribute to Candida colonization resistance. Understanding fungal-epithelial interactions in mammalian models of disease is critical to predict vulnerabilities and to identify opportunities for immune-based strategies to treat fungal infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Abundant evidence suggests that chronic inflammation is linked to prostate cancer and that infection is a possible cause of prostate cancer., Methods: To identify microbiota or pathogens associated with prostate cancer, we investigated the transcriptomes of 20 human prostate cancer tissues. We performed de novo assembly of nonhuman sequences from RNA-seq data., Results: We identified four bacteria as candidate microbiota in the prostate, including Moraxella osloensis, Uncultured chroococcidiopsis, Cutibacterium acnes, and Micrococcus luteus. Among these, C. acnes was detected in 19 of 20 prostate cancer tissue samples by immunohistochemistry. We then analyzed the gene expression profiles of prostate epithelial cells infected in vitro with C. acnes and found significant changes in homologous recombination (HR) and the Fanconi anemia pathway. Notably, electron microscopy demonstrated that C. acnes invaded prostate epithelial cells and localized in perinuclear vesicles, whereas analysis of γH2AX foci and HR assays demonstrated impaired HR repair. In particular, BRCA2 was significantly downregulated in C. acnes-infected cells., Conclusions: These findings suggest that C. acnes infection in the prostate could lead to HR deficiency (BRCAness) which promotes DNA double-strand breaks, thereby increasing the risk of cancer development., (© 2024 Wiley Periodicals LLC.)

Bacillus species, which have two cell-type forms (vegetative cells and spores), demonstrate a variety of probiotic functions in animal feed additives and human nutrition. We previously found that the probiotic effect of Bacillus subtilis S-2 spores with high germination response to L-alanine was specifically enhanced by the L-alanine pretreatment. The germination response of Bacillus is highly associated with the germination receptors of spores. However, how L-alanine-induced germination of spores exerts anti-infectious effect in epithelial cells remains unclear. In this study, we constructed the mutant strain of B. subtilis S-2 with germination receptor gerAA knockout to further explore the role of spore germination in resisting pathogen infection to cells. The differential probiotic effects of B. subtilis S-2 and S-2 ΔgerAA spores pretreated with L-alanine were evaluated in intestinal porcine epithelial cells (IPEC-J2) or Caco2 cells infected with enterotoxigenic Escherichia coli (ETEC) or following IL-1β stimulation. The results showed that the germination response of the S-2 ΔgerAA spores to L-alanine was significantly reduced. Compared with the S-2 ΔgerAA spores, the L-alanine-induced germination of B. subtilis S-2 spores significantly increased the activity of anti-adhesion of ETEC to IPEC-J2 cells and reduced the expression of inflammatory factors and cell receptors. L-alanine induction also significantly promoted the expression of autophagy-related proteins in the B. subtilis S-2 spores. These findings demonstrate that the gerAA germination receptor is essential for the probiotic function of Bacillus spores and that L-alanine treatment promotes the anti-infectious properties of the germinated spores in porcine intestinal epithelial IPEC-J2 cells. The result suggests the importance of germination receptor gerAA in helping spore germination and enhancing anti-infectious activity. The findings in the study benefit to screening of potential Bacillus probiotics and increasing probiotic efficacy induced by L-alanine as an adjuvant., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Helicobacter pylori infection is the major risk factor associated with the development of gastric cancer. Currently, administration of standard antibiotic therapy combined with probiotics and postbiotics has gained significant attention in the management of H. pylori infection. In this work, the immunomodulatory effects of Lactobacillus crispatus-derived extracellular vesicles (EVs) and cell-free supernatant (CFS) were investigated on H. pylori-induced inflammatory response in human gastric adenocarcinoma (AGS) cells. L. crispatus-derived EVs were isolated by ultracentrifugation and physically characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Furthermore, the protein content of L. crispatus-derived EVs was also evaluated by SDS-PAGE. Cell viability of AGS cells exposed to varying concentrations of EVs and CFS was assessed by MTT assay. The mRNA expression of IL-1β, IL-6, IL-8, TNF-α, IL-10, and TGF-ß genes was determined by RT-qPCR. ELISA was used for the measurement of IL-8 production in AGS cells. In addition, EVs (50 μg/mL) and CFS modulated the H. pylori-induced inflammation by downregulating the mRNA expression of IL-1β, IL-6, IL-8, and TNF-α, and upregulating the expression of IL-10, and TGF-ß genes in AGS cells. Furthermore, H. pylori-induced IL-8 production was dramatically decreased after treatment with L. crispatus-derived EVs and CFS. In conclusion, our observation suggests for the first time that EVs released by L. crispatus strain RIGLD-1 and its CFS could be recommended as potential therapeutic agents against H. pylori-triggered inflammation., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Arcobacter butzleri is a foodborne pathogen that mainly causes enteritis in humans, but the number of cases of bacteraemia has increased in recent years. However, there is still limited knowledge on the pathogenic mechanisms of this bacterium. To investigate how A. butzleri causes disease, single knockout mutants were constructed in the cadF, ABU&#95;RS00335, ciaB, and flaAB genes, which might be involved in adhesion and invasion properties. These mutants and the isogenic wild-type (WT) were then tested for their ability to adhere and invade human Caco-2 and HT29-MTX cells. The adhesion and invasion of A. butzleri RM4018 strain was also visualized by a Leica CTR 6500 confocal microscope. The adhesion and invasion abilities of mutants lacking the invasion antigen CiaB or a functional flagellum were lower than those of the WTs. However, the extent of the decrease varied depending on the strain and/or cell line. Mutants lacking the fibronectin (FN)-binding protein CadF consistently exhibited reduced abilities, while the inactivation of the other studied FN-binding protein, ABU&#95;RS00335, led to a reduction in only one of the two strains tested. Therefore, the ciaB and flaAB genes appear to be important for A. butzleri adhesion and invasion properties, while cadF appears to be indispensable., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Itsaso Baztarrika Uria reports financial support was provided by Basque Government. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

B. parapertussis is a bacterium that causes whooping cough, a severe respiratory infection disease, that has shown an increased incidence in the population. Upon transmission through aerosol droplets, the initial steps of host colonization critically depend on the bacterial adhesins. We here described BPP0974, a B. parapertussis protein that exhibits the typical domain architecture of the large repetitive RTX adhesin family. BPP0974 was found to be retained in the bacterial membrane and secreted into the culture medium. This protein was found overexpressed in the avirulent phase of B. parapertussis, the phenotype proposed for initial host colonization. Interestingly, BPP0974 was found relevant for the biofilm formation as well as involved in the bacterial attachment to and survival within the respiratory epithelial cells. Taken together, our results suggest a role for BPP0974 in the early host colonization and pathogenesis of B. parapertussis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Helicobacter pylori ( H. pylori ) infection is the primary risk factor for the progress of gastric diseases. The persistent stomach colonization of H. pylori is closely associated with the development of gastritis and malignancies. Although the involvement of progranulin (PGRN) in various cancer types has been well-documented, its functional role and underlying mechanisms in gastric cancer (GC) associated with H. pylori infection remain largely unknown. This report demonstrated that PGRN was up-regulated in GC and associated with poor prognosis, as determined through local and public database analysis. Additionally, H. pylori induced the up-regulation of PGRN in gastric epithelial cells both in vitro and in vivo . Functional studies have shown that PGRN promoted the intracellular colonization of H. pylori . Mechanistically, H. pylori infection induced autophagy, while PGRN inhibited autophagy to promote the intracellular colonization of H. pylori . Furthermore, PGRN suppressed H. pylori -induced autophagy by down-regulating decorin (DCN) through the mTOR pathway. In general, PGRN inhibited autophagy to facilitate intracellular colonization of H. pylori via the PGRN/mTOR/DCN axis. This study provides new insights into the molecular mechanisms underlying the progression of gastric diseases, suggesting PGRN as a potential therapeutic target and prognostic predictor for these disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Xiang, Zhou, Ren, Shi, Du, Fu, Li and Wang.)

Numerous Aspergillus fumigatus (Af) airborne spores are inhaled daily by humans and animals due to their ubiquitous presence. The interaction between the spores and the respiratory epithelium, as well as its impact on the epithelial barrier function, remains largely unknown. The epithelial barrier protects the respiratory epithelium against viral infections. However, it can be compromised by environmental contaminants such as pollen, thereby increasing susceptibility to respiratory viral infections, including alphaherpesvirus equine herpesvirus type 1 (EHV-1). To determine whether Af spores disrupt the epithelial integrity and enhance susceptibility to viral infections, equine respiratory mucosal ex vivo explants were pretreated with Af spore diffusate, followed by EHV-1 inoculation. Spore proteases were characterized by zymography and identified using mass spectrometry-based proteomics. Proteases of the serine protease, metalloprotease, and aspartic protease groups were identified. Morphological analysis of hematoxylin-eosin (HE)-stained sections of the explants revealed that Af spores induced the desquamation of epithelial cells and a significant increase in intercellular space at high and low concentrations, respectively. The increase in intercellular space in the epithelium caused by Af spore proteases correlated with an increase in EHV-1 infection. Together, our findings demonstrate that Af spore proteases disrupt epithelial integrity, potentially leading to increased viral infection of the respiratory epithelium.

The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) has led to the development of novel anti-tuberculosis (anti-TB) drugs. Common methods for testing the efficacy of new drugs, including two-dimensional cell culture models or animal models, have several limitations. Therefore, an appropriate model representative of the human organism is required. Here, we developed an M.tb infection model using human lung organoids (hLOs) and demonstrated that M.tb H37Rv can infect lung epithelial cells and human macrophages (hMφs) in hLOs. This novel M.tb infection model can be cultured long-term and split several times while maintaining a similar number of M.tb H37Rv inside the hLOs. Anti-TB drugs reduced the intracellular survival of M.tb in hLOs. Notably, M.tb growth in hLOs was effectively suppressed at each passage by rifampicin and bedaquiline. Furthermore, a reduction in inflammatory cytokine production and intracellular survival of M.tb were observed upon knockdown of MFN2 and HERPUD1 (host-directed therapeutic targets for TB) in our M.tb H37Rv-infected hLO model. Thus, the incorporation of hMφs and M.tb into hLOs provides a powerful strategy for generating an M.tb infection model. This model can effectively reflect host-pathogen interactions and be utilized to test the efficacy of anti-TB drugs and host-directed therapies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Edwardsiella ictaluri is a Gram-negative, facultative intracellular bacterium that causes enteric septicemia in catfish (ESC). The RNA chaperone Hfq (host factor for phage Qβ replication) facilitates gene regulation via small RNAs (sRNAs) in various pathogenic bacteria. Despite its significance in other bacterial species, the role of hfq in E. ictaluri remains unexplored. This study aimed to elucidate the role of hfq in E. ictaluri by creating an hfq mutant ( Ei Δ hfq ) through in-frame gene deletion and characterization. Our findings revealed that the Hfq protein is highly conserved within the genus Edwardsiella . The deletion of hfq resulted in a significantly reduced growth rate during the late exponential phase. Additionally, Ei Δ hfq displayed a diminished capacity for biofilm formation and exhibited increased motility. Under acidic and oxidative stress conditions, Ei Δ hfq demonstrated impaired growth, and we observed elevated hfq expression when subjected to in vitro and in vivo stress conditions. Ei Δ hfq exhibited reduced survival within catfish peritoneal macrophages, although it had no discernible effect on the adherence and invasion of epithelial cells. The infection model revealed that hfq is needed for bacterial persistence in catfish, and its absence caused significant virulence attenuation in catfish. Finally, the Ei Δ hfq vaccination completely protected catfish against subsequent Ei WT infection. In summary, these results underscore the pivotal role of hfq in E. ictaluri , affecting its growth, motility, biofilm formation, stress response, and virulence in macrophages and within catfish host., Competing Interests: The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Akgul, Kalindamar, Kordon, Abdelhamed, Ibrahim, Tekedar and Karsi.)

Introduction: Bacterial urinary tract infections (UTI) are among the most common infectious diseases worldwide. The rise of multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) UTI cases is a significant threat to healthcare systems. Several probiotic bacteria have been proposed as an alternative to combat MDR UTI. Lactic acid bacteria in the genus Limosilactobacillus are some of the most studied and used probiotics. However, strain-specific effects play a critical role in probiotic properties. L. reuteri KUB-AC5 (AC5), isolated from the chicken gut, confers antimicrobial and immunobiotic effects against some human pathogens. However, the antibacterial and immune modulatory effects of AC5 on UPEC have never been explored., Methods: Here, we investigated both the direct and indirect effects of AC5 against UPEC isolates (UTI89, CFT073, and clinical MDR UPEC AT31) in vitro . Using a spot-on lawn, agar-well diffusion, and competitive growth assays, we found that viable AC5 cells and cell-free components of this probiotic significantly reduced the UPEC growth of all strains tested. The human bladder epithelial cell line UM-UC-3 was used to assess the adhesion and pathogen-attachment inhibition properties of AC5 on UPEC., Results and Discussion: Our data showed that AC5 can attach to UM-UC-3 and decrease UPEC attachment in a dose-dependent manner. Pretreatment of UPEC-infected murine macrophage RAW264.7 cells with viable AC5 (multiplicity of infection, MOI = 1) for 24 hours enhanced macrophage-killing activity and increased proinflammatory ( Nos2 , Il6 , and Tnfa ) and anti-inflammatory ( Il10 ) gene expression. These findings indicate the gut-derived AC5 probiotic could be a potential urogenital probiotic against MDR UTI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tantibhadrasapa, Li, Buddhasiri, Sukjoi, Mongkolkarvin, Boonpan, Wongpalee, Paenkaew, Sutheeworapong, Nakphaichit, Nitisinprasert, Hsieh and Thiennimitr.)

Indole-3-lactic acid (ILA) has exhibited antimicrobial properties. However, its role in inhibiting Helicobacter pylori infection remains elusive. This study investigated the inhibitory effect of ILA produced by Lacticaseibacillus paracasei on H. pylori , which was further confirmed by cell and animal experiments. 5 mg/mL ILA was sufficient to directly inhibit the growth of H. pylori in vitro, with a urease inhibitory activity reaching 60.94 ± 1.03%, and the cell morphology and structure were destroyed. ILA inhibited 56.5% adhesion of H. pylori to GES-1 and significantly reduced the number of apoptotic cells. Furthermore, ILA suppresses H. pylori colonization by approximately 38% to 63%, reduced inflammation and oxidative stress in H. pylori -infected mice, and enhanced the enrichment and variety of gut microbiota, notably fostering the growth of beneficial bacteria such as Lactobacillus and Bifidobacterium strains. The results support that ILA derived from Lactobacillus can be applicated as a novel prebiotic in anti- H. pylori functional foods.

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this, the molecular mechanisms that allow H. influenzae to establish persistent infections of human epithelia are not well understood. Here, we have investigated how H. influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, 'unproductive' inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extracellular and intraepithelial burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations, and appears to involve interruption of NFκB signalling. This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been observed, and we were able to demonstrate that only infections with live, but not heat-killed H. influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1β, IL-36γ and TNFα. Interestingly, NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection, resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H. influenzae in the host, our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kappler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Respiratory infections caused by the human fungal pathogen Aspergillus fumigatus are a major cause of mortality for immunocompromised patients. Exposure to these pathogens occurs through inhalation, although the role of the respiratory epithelium in disease pathogenesis has not been fully defined. Employing a primary human airway epithelial model, we demonstrate that fungal melanins potently block the post-translational secretion of the chemokines CXCL1 and CXCL8 independent of transcription or the requirement of melanin to be phagocytosed, leading to a significant reduction in neutrophil recruitment to the apical airway both in vitro and in vivo. Aspergillus-derived melanin, a major constituent of the fungal cell wall, dampened airway epithelial chemokine secretion in response to fungi, bacteria, and exogenous cytokines. Furthermore, melanin muted pathogen-mediated calcium fluxing and hindered actin filamentation. Taken together, our results reveal a critical role for melanin interaction with airway epithelium in shaping the host response to fungal and bacterial pathogens., (© 2024. The Author(s).)

Brucella abortus (Ba) is a pathogen that survives inside macrophages. Despite being its preferential niche, Ba infects other cells, as shown by the multiple signs and symptoms humans present. This pathogen can evade our immune system. Ba displays a mechanism of down-modulating MHC-I on monocytes/macrophages in the presence of IFN-γ (when Th1 response is triggered) without altering the total expression of MHC-I. The retained MHC-I proteins are located within the Golgi Apparatus (GA). The RNA of Ba is one of the PAMPs that trigger this phenomenon. However, we acknowledged whether this event could be triggered in other cells relevant during Ba infection. Here, we demonstrate that Ba RNA reduced the surface expression of MHC-I induced by IFN-γ in the human bronchial epithelium (Calu-6), the human alveolar epithelium (A-549) and the endothelial microvasculature (HMEC) cell lines. In Calu-6 and HMEC cells, Ba RNA induces the retention of MHC-I in the GA. This phenomenon was not observed in A-549 cells. We then evaluated the effect of Ba RNA on the secretion of IL-8, IL-6 and MCP-1, key cytokines in Ba infection. Contrary to our expectations, HMEC, Calu-6 and A-549 cells treated with Ba RNA had higher IL-8 and IL-6 levels compared to untreated cells. In addition, we showed that Ba RNA down-modulates the MHC-I surface expression induced by IFN-γ on human monocytes/macrophages via the pathway of the Epidermal Growth Factor Receptor (EGFR). So, cells were stimulated with an EGFR ligand-blocking antibody (Cetuximab) and Ba RNA. Neutralization of the EGFR to some extent reversed the down-modulation of MHC-I mediated by Ba RNA in HMEC and A-549 cells. In conclusion, this is the first study exploring a central immune evasion strategy, such as the downregulation of MHC-I surface expression, beyond monocytes and could shed light on how it persists effectively within the host, enduring unseen and escaping CD8+ T cell surveillance., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Serafino et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The human intestinal tract is colonized with microorganisms, which present a diverse array of immunological challenges. A number of antimicrobial mechanisms have evolved to cope with these challenges. A key defense mechanism is the expression of inducible antimicrobial peptides (AMPs), such as beta-defensins, which rapidly inactivate microorganisms. We currently have a limited knowledge of mechanisms regulating the inducible expression of AMP genes, especially factors from the host required in these regulatory mechanisms. To identify the host factors required for expression of the beta-defensin-2 gene (HBD2) in intestinal epithelial cells upon a bacterial challenge, we performed a RNAi screen using a siRNA library spanning the whole human genome. The screening was performed in duplicate to select the strongest 79 and 110 hit genes whose silencing promoted or inhibited HBD2 expression, respectively. A set of 57 hits selected among the two groups of genes was subjected to a counter-screening and a subset was subsequently validated for its impact onto HBD2 expression. Among the 57 confirmed hits, we brought out the TLR5-MYD88 signaling pathway, but above all new signaling proteins, epigenetic regulators and transcription factors so far unrevealed in the HBD2 regulatory circuits, like the GATA6 transcription factor involved in inflammatory bowel diseases. This study represents a significant step toward unveiling the key molecular requirements to promote AMP expression in human intestinal epithelial cells, and revealing new potential targets for the development of an innovative therapeutic strategy aiming at stimulating the host AMP expression, at the era of antimicrobial resistance., (© 2024. The Author(s).)

Epithelial cells are the first point of contact for bacteria entering the respiratory tract. Streptococcus pneumoniae is an obligate human pathobiont of the nasal mucosa, carried asymptomatically but also the cause of severe pneumoniae. The role of the epithelium in maintaining homeostatic interactions or mounting an inflammatory response to invasive S. pneumoniae is currently poorly understood. However, studies have shown that chromatin modifications, at the histone level, induced by bacterial pathogens interfere with the host transcriptional program and promote infection. Here, we uncover a histone modification induced by S. pneumoniae infection maintained for at least 9 days upon clearance of bacteria with antibiotics. Di-methylation of histone H3 on lysine 4 (H3K4me2) is induced in an active manner by bacterial attachment to host cells. We show that infection establishes a unique epigenetic program affecting the transcriptional response of epithelial cells, rendering them more permissive upon secondary infection. Our results establish H3K4me2 as a unique modification induced by infection, distinct from H3K4me3 or me1, which localizes to enhancer regions genome-wide. Therefore, this study reveals evidence that bacterial infection leaves a memory in epithelial cells after bacterial clearance, in an epigenomic mark, thereby altering cellular responses to subsequent infections and promoting infection., (© 2024. The Author(s).)

Mycoplasma bovis mastitisis highly contagious and disrupts lactation, posing a significant threat to the dairy industry. While the mammary gland's defence mechanism involves epithelial cells and mononuclear cells (MNC), their interaction with M. bovis remains incompletely understood. In this study, we assessed the immunological reactivity of bovine mammary epithelial cells (bMEC) to M. bovis through co-culture with MNC. Upon co-culture with MNC, the mRNA expression levels of interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α in bMEC stimulated by M. bovis showed a significant increase compared to monoculture. Additionally, when stimulated with M. bovis, the culture supernatant exhibited significantly higher concentrations of IL-6 and interferon (IFN)-γ, while IL-1β concentration tended to be higher in co-culture with MNC than in monoculture. Furthermore, the mRNA expression levels of toll-like receptor (TLR) 2 in bMEC stimulated with M. bovis tended to increase, and TLR4 significantly increased when co-cultured with MNC compared to monocultures. However, the surface expression levels in bMEC did not exhibit significant changes between co-culture and monoculture. Overall, our research indicates that the inflammatory response of bMEC is increased during co-culture with MNC, suggesting that the interaction between bMEC and MNC in the mammary gland amplifies the immune response to M. bovis in cows affected by M. bovis mastitis.

Background: Helicobacter pylori infection-associated gastric adenocarcinoma is influenced by various factors, including the digestive microbiota. Lactic acid bacteria role in digestive carcinogenesis has been discussed, and some Lactobacillaceae family species have been shown to act against H. pylori-induced inflammation and colonization. However, their effects on H. pylori-related carcinogenesis have not yet been studied. Lactobacillaceae family effects on the epithelial-to-mesenchymal transition (EMT), emergence of cells with cancer stem cell (CSC) properties and the pro-inflammatory response of gastric epithelial cells to H. pylori infection were investigated., Materials and Methods: A co-culture model of AGS gastric epithelial cells infected with a carcinogenic strain of H. pylori associated with 18 different probiotic strains candidates were used. Different EMT indicators and CSC properties were studied, including quantification of the mesenchymal phenotype, tumorsphere formation, EMT marker expression, and tight junction evaluation with immunofluorescence microscopy. The effect of the strains on the pro-inflammatory response to H. pylori was also evaluated by quantifying interleukin-8 (IL-8) production using ELISA., Results: Among the strains tested, Lactobacillus gasseri BIO6369 and Lacticaseibacillus rhamnosus BIO5326 induced a 30.6% and 38.4% reduction in the mesenchymal phenotype, respectively, caused a significant decrease in Snail and Zeb1 EMT marker expression and prevented the loss of tight junctions induced by H. pylori infection. A separate co-culture with a Boyden chamber maintained the effects induced by the two strains. H. pylori-induced IL-8 production was also significantly reduced in the presence of L. gasseri BIO6369 and L. rhamnosus BIO5326., Conclusion: Lactobacillus gasseri BIO6369 and L. rhamnosus BIO5326 strains decreased epithelial-to-mesenchymal transition and inflammation induced by H. pylori infection, suggesting that these species may have a protective effect against H. pylori-induced gastric carcinogenesis., (© 2024 The Author(s). Helicobacter published by John Wiley & Sons Ltd.)

Structural changes to the vocal fold (VF) epithelium, namely, loosened intercellular junctions, have been reported in VF benign lesions. The potential mechanisms responsible for the disruption of cell junctions do not address the contribution of resident microbial communities to this pathological phenomenon. In this study, we focused on determining the relationship between Streptococcus pseudopneumoniae (SP), a dominant bacterial species associated with benign lesions, and Streptococcus salivarius (SS), a commensal bacterium, with human VF epithelial cells in our three-dimensional model of the human VF mucosa. This experimental system enabled direct deposition of bacteria onto constructs at the air/liquid interface, allowing for the assessment of bacterium-host interactions at the cellular, molecular and ultrastructural levels. Our findings demonstrate that SP disrupts VF epithelial integrity and initiates inflammation via the exported products HtrA1 and pneumolysin. In contrast, SS attaches to the VF epithelium, reduces inflammation and induces Mmp2-mediated apical desquamation of infected cells to mitigate the impact of pathogens. In conclusion, this study highlights the complexity of microbial involvement in VF pathology and potential VF mucosal restoration in the presence of laryngeal commensals., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Glaesserella parasuis (G. parasuis) is the causative agent of porcine Glässer's disease, resulting in high mortality rates in pigs due to excessive inflammation-induced tissue damage. Previous studies investigating the protective effects of G. parasuis vaccination indicated a possible role of ApoA1 in reflecting disease progression following G. parasuis infection. However, the mechanisms of ApoA1 expression and its role in these infections are not well understood. In this investigation, newborn porcine tracheal (NPTr) epithelial cells infected with G. parasuis were used to elucidate the molecular mechanism and role of ApoA1. The study revealed that the AMPK pathway activation inhibited ApoA1 expression in NPTr cells infected with G. parasuis for the first time. Furthermore, Egr1 was identified as a core transcription factor regulating ApoA1 expression using a CRISPR/Cas9-based system. Importantly, it was discovered that APOA1 protein significantly reduced apoptosis, pyroptosis, necroptosis, and inflammatory factors induced by G. parasuis in vivo. These findings not only enhance our understanding of ApoA1 in response to bacterial infections but also highlight its potential in mitigating tissue damage caused by G. parasuis infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The oral cavity, a unique ecosystem harboring diverse microorganisms, maintains health through a balanced microflora. Disruption may lead to disease, emphasizing the protective role of gingival epithelial cells (GECs) in preventing harm from pathogenic oral microbes. Shifting GECs' response from proinflammatory to antimicrobial could be a novel strategy for periodontitis. Photobiomodulation therapy (PBMT), a nonpharmacologic host modulatory approach, is considered an alternative to drugs. While the host cell response induced by a single type of pathogen-associated molecular patterns (PAMPs) was widely studied, this model does not address the cellular response to intact microbes that exhibit multiple PAMPs that might modulate the response. Inspired by this, we developed an in vitro model that simulates direct interactions between host cells and intact pathogens and evaluated the effect of PBMT on the response of human gingival keratinocytes (HGKs) to challenge viable oral microbes at both the cellular and molecular levels. Our data demonstrated that LED pretreatment on microbially challenged HGKs with specific continuous wavelengths (red: 615 nm; near-infrared: 880 nm) induced the production of various antimicrobial peptides, enhanced cell viability and proliferation, promoted reactive oxygen species scavenging, and down-modulated proinflammatory activity. The data also suggest a potential explanation regarding the superior efficacy of near-infrared light treatment compared with red light in enhancing antimicrobial activity and reducing cellular inflammation of HGKs. Taken together, the findings suggest that PBMT enhances the overall barrier function of gingival epithelium while minimizing inflammation-mediated breakdown of the underlying structures., Competing Interests: Declaration of Conflicting InterestsThe authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: G.H. and A.K. are co-founders of LEDEN LLC. G.H., A.K., J.K., and H.-E.K. are inventors on US Patent Application No. 18/064,747 (filed on December 12, 2022).

Vulvovaginal candidiasis (VVC) is a prevalent condition affecting a significant portion of women worldwide. Licochalcone A (LA), a natural compound with diverse biological activities, holds promise as a protective agent against Candida albicans (C. albicans) infection. This study aims to investigate the potential of LA to safeguard vaginal epithelial cells (VECs) from C. albicans infection and elucidate the underlying molecular mechanisms. To simulate VVC in vitro, VK2-E6E7 cells were infected with C. albicans. Candida albicans biofilm formation, C. albicans adhesion to VK2-E6E7 cells, and C. albicans-induced cell damage and inflammatory responses were assessed by XTT reduction assay, fluorescence assay, LDH assay, and ELISA. CCK-8 assay was performed to evaluate the cytotoxic effects of LA on VK2-E6E7 cells. Western blotting assay was performed to detect protein expression. LA dose-dependently hindered C. albicans biofilm formation and adhesion to VK2-E6E7 cells. Furthermore, LA mitigated cell damage, inhibited the Bax/Bcl-2 ratio, and attenuated the secretion of pro-inflammatory cytokines in C. albicans-induced VK2-E6E7 cells. The investigation into LA's impact on the Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway revealed that LA downregulated TLR4 expression and inhibited NF-κB activation in C. albicans-infected VK2-E6E7 cells. Furthermore, TLR4 overexpression partially abated LA-mediated protection, further highlighting the role of the TLR4/NF-κB pathway. LA holds the potential to safeguard VECs against C. albicans infection, potentially offering therapeutic avenues for VVC management., (© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Heme oxygenase HO-1 (HMOX) regulates cellular inflammation and apoptosis, but its role in regulation of autophagy in Mycoplasma bovis infection is unknown. The objective was to determine how the HO-1/CO- Protein kinase RNA-like endoplasmic reticulum kinase (PERK)-Ca 2+ - transcription factor EB (TFEB) signaling axis induces autophagy and regulates clearance of M. bovis by bovine mammary epithelial cells (bMECs). M. bovis inhibited autophagy and lysosomal biogenesis in bMECs and suppressed HO-1 protein and expression of related proteins, namely nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (keap1). Activation of HO-1 and its production of carbon monoxide (CO) were required for induction of autophagy and clearance of intracellular M. bovis. Furthermore, when HO-1 was deficient, CO sustained cellular autophagy. HO-1 activation increased intracellular calcium (Ca 2+ ) and cytosolic localization activity of TFEB via PERK. Knockdown of PERK or chelation of intracellular Ca 2+ inhibited HO-1-induced M. bovis autophagy and clearance. M. bovis infection affected nuclear localization of lysosomal TFEB in the MiT/TFE transcription factor subfamily, whereas activation of HO-1 mediated dephosphorylation and intranuclear localization of TFEB, promoting autophagy, lysosomal biogenesis and autophagic clearance of M. bovis. Nuclear translocation of TFEB in HO-1 was critical to induce M. bovis transport and survival of infected bMECs. Furthermore, the HO-1/CO-PERK-Ca 2+ -TFEB signaling axis induced autophagy and M. bovis clearance, providing a viable approach to treat persistent M. bovis infections., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The human pathogenic fungus Candida albicans damages epithelial cells during superficial infections. Here we use three-dimensional-sequential-confocal Raman spectroscopic imaging and atomic force microscopy to investigate the interaction of C. albicans wild type cells, the secreted C. albicans peptide toxin candidalysin and mutant cells lacking candidalysin with epithelial cells. The candidalysin is responsible for epithelial cell damage and exhibits in its deuterated form an identifiable Raman signal in a frequency region distinct from the cellular frequency region. Vibration modes at 2100-2200 cm -1 attributed to carbon‑deuterium bending and at 477 cm -1 , attributed to the nitrogen‑deuterium out-of-plane bending, found around the nucleus, can be assigned to deuterated candidalysin. Atomic force microscopy visualized 100 nm deep lesions on the cell and force-distance curves indicate the higher adhesion on pore surrounding after incubation with candidalysin. Candidalysin targets the plasma membrane, but is also found inside of the cytosol of epithelial cells during C. albicans infection., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Glaesserella parasuis (G. parasuis) is a common Gram-negative commensal bacterium in the upper respiratory tract of swine that can cause Glässer's disease under stress conditions. Pyroptosis is an important immune defence mechanism of the body that plays a crucial role in clearing pathogen infections and endogenous danger signals. This study aimed to investigate the mechanism of G. parasuis serotype 5 SQ (GPS5-SQ)-induced pyroptosis in swine tracheal epithelial cells (STECs). The results of the present study demonstrated that GPS5-SQ infection induces pyroptosis in STECs by enhancing the protein level of the N-terminal domain of gasdermin D (GSDMD-N) and activating the NOD-like receptor protein 3 (NLRP3) inflammasome. Furthermore, the levels of pyroptosis-related proteins, including GSDMD-N and cleaved caspase-1 were considerably decreased in STECs after the knockdown of retinoic acid inducible gene-I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). These results indicated that GPS5-SQ might trigger pyroptosis through the activation of the RIG-I/MAVS/NLRP3 signaling pathway. More importantly, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) repressed the activation of the RIG-I/MAVS/NLRP3 signaling and rescued the decrease in Occludin and zonula occludens-1 (ZO-1) after GPS5-SQ infection. Overall, our findings show that GPS5-SQ can activate RIG-I/MAVS/NLRP3 signaling and destroy the integrity of the epithelial barrier by inducing ROS generation in STECs, shedding new light on G. parasuis pathogenesis., Competing Interests: Declaration of Competing Interest The authors declare they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Salmonella enterica subspecies enterica serovar Typhimurium is an intracellular pathogen that invades and colonizes the intestinal epithelium. Following bacterial invasion, Salmonella is enclosed within a membrane-bound vacuole known as a Salmonella -containing vacuole (SCV). However, a subset of Salmonella has the capability to prematurely rupture the SCV and escape, resulting in Salmonella hyper-replication within the cytosol of epithelial cells. A recently published RNA-seq study provides an overview of cytosolic and vacuolar upregulated genes and highlights pagN vacuolar upregulation. Here, using transcription kinetics, protein production profile, and immunofluorescence microscopy, we showed that PagN is exclusively produced by Salmonella in SCV. Gentamicin protection and chloroquine resistance assays were performed to demonstrate that deletion of pagN affects Salmonella replication by affecting the cytosolic bacterial population. This study presents the first example of a Salmonella virulence factor expressed within the endocytic compartment, which has a significant impact on the dynamics of Salmonella cytosolic hyper-replication.

Helicobacter pylori is a highly prevalent human gastric pathogen that causes gastritis, ulcer disease, and gastric cancer. It is not yet fully understood how H. pylori injures the gastric epithelium. The Na,K-ATPase, an essential transporter found in virtually all mammalian cells, has been shown to be important for maintaining the barrier function of lung and kidney epithelia. H. pylori decreases levels of Na,K-ATPase in the plasma membrane of gastric epithelial cells, and the aim of this study was to demonstrate that this reduction led to gastric injury by impairing the epithelial barrier. Similar to H. pylori infection, the inhibition of Na,K-ATPase with ouabain decreased transepithelial electrical resistance and increased paracellular permeability in cell monolayers of human gastric cultured cells, 2D human gastric organoids, and gastric epithelium isolated from gerbils. Similar effects were caused by a partial shRNA silencing of Na,K-ATPase in human gastric organoids. Both H. pylori infection and ouabain exposure disrupted organization of adherens junctions in human gastric epithelia as demonstrated by E-cadherin immunofluorescence. Functional and structural impairment of epithelial integrity with a decrease in Na,K-ATPase amount or activity provides evidence that the H. pylori -induced downregulation of Na,K-ATPase plays a role in the complex mechanism of gastric disease induced by the bacteria.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's Disease, a chronic granulomatous enteritis of ruminants. MAP establishes an infection in the host via the small intestine. This requires the bacterium to adhere to, and be internalised by, cells of the intestinal tract. The effector molecules expressed by MAP for this purpose remain to be fully identified and understood. Mammalian cell entry (mce) proteins have been shown to enable other Mycobacterial species to attach to and invade host epithelial cells. Here, we have expressed Mce1A, Mce1D, Mce3C and Mce4A proteins derived from MAP on the surface of a non-invasive Escherichia coli to characterise their role in the initial interaction between MAP and the host. To this end, expression of mce1A was found to significantly increase the ability of the E. coli to attach and survive intracellularly in human monocyte-like THP-1 cells, whereas expression of mce1D was found to significantly increase attachment and invasion of E. coli to bovine epithelial cell-like MDBK cells, implying cell-type specificity. Furthermore, expression of Mce1A and Mce1D on the surface of a previously non-invasive E. coli enhanced the ability of the bacterium to infect 3D bovine basal-out enteroids. Together, our data contributes to our understanding of the effector molecules utilised by MAP in the initial interaction with the host, and may provide potential targets for therapeutic intervention., (© 2024. The Author(s).)

Impaired E-cadherin (Cdh1) functions are closely associated with cellular dedifferentiation, infiltrative tumor growth and metastasis, particularly in gastric cancer. The class-I carcinogen Helicobacter pylori ( H. pylori ) colonizes gastric epithelial cells and induces Cdh1 shedding, which is primarily mediated by the secreted bacterial protease high temperature requirement A (HtrA). In this study, we used human primary epithelial cell lines derived from gastroids and mucosoids from different healthy donors to investigate HtrA-mediated Cdh1 cleavage and the subsequent impact on bacterial pathogenesis in a non-neoplastic context. We found a severe impairment of Cdh1 functions by HtrA-induced ectodomain cleavage in 2D primary cells and mucosoids. Since mucosoids exhibit an intact apico-basal polarity, we investigated bacterial transmigration across the monolayer, which was partially depolarized by HtrA, as indicated by microscopy, the analyses of the transepithelial electrical resistance (TEER) and colony forming unit (cfu) assays. Finally, we investigated CagA injection and observed efficient CagA translocation and tyrosine phosphorylation in 2D primary cells and, to a lesser extent, similar effects in mucosoids. In summary, HtrA is a crucially important factor promoting the multistep pathogenesis of H. pylori in non-transformed primary gastric epithelial cells and organoid-based epithelial models., Competing Interests: The authors declare no conflicts of interest.