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Many bacterial pathogens have evolved effective strategies to interfere with the ubiquitination network to evade clearance by the innate immune system. Here, we report that OTUB1, one of the most abundant deubiquitinases (DUBs) in mammalian cells, is subjected to both canonical and noncanonical ubiquitination during Legionella pneumophila infection. The effectors SidC and SdcA catalyze OTUB1 ubiquitination at multiple lysine residues, resulting in its association with a Legionella-containing vacuole. Lysine ubiquitination by SidC and SdcA promotes interactions between OTUB1 and DEPTOR, an inhibitor of the MTORC1 pathway, thus suppressing MTORC1 signaling. The inhibition of MTORC1 leads to suppression of host protein synthesis and promotion of host macroautophagy/autophagy during L. pneumophila infection. In addition, members of the SidE family effectors (SidEs) induce phosphoribosyl (PR)-linked ubiquitination of OTUB1 at Ser16 and Ser18 and block its DUB activity. The levels of the lysine and serine ubiquitination of OTUB1 are further regulated by effectors that function to antagonize the activities of SidC, SdcA and SidEs, including Lem27, DupA, DupB, SidJ and SdjA. Our study reveals an effectors-mediated complicated mechanism in regulating the activity of a host DUB. Abbreviations: BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; DUB: deubiquitinase; Dot/Icm: defective for organelle trafficking/intracellular multiplication; DEPTOR: DEP domain containing MTOR interacting protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L. pneumophila: Legionella pneumophila; LCV: Legionella-containing vacuole; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; OTUB1: OTU deubiquitinase, ubiquitin aldehyde binding 1; PR-Ub: phosphoribosyl (PR)-linked ubiquitin; PTM: posttranslational modification; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SidEs: SidE family effectors; Ub: ubiquitin. [ABSTRACT FROM AUTHOR]

Chlamydiae are bacteria that are intriguing and important at the same time. The genus Chlamydia encompasses many species of obligate intracellular organisms: they can multiply only inside the cells of their host organism. Many, perhaps most animals have their own specifically adapted chlamydial species. In humans, the clinically most relevant species is Chlamydia trachomatis, which has particular importance as an agent of sexually transmitted disease. Pigs are the natural host of Chlamydia suis but may also carry Chlamydia abortus and Chlamydia pecorum. C. abortus and possibly C. suis have anthropozoonotic potential, which makes them interesting to human medicine, but all three species bring a substantial burden of disease to pigs. The recent availability of genomic sequence comparisons suggests adaptation of chlamydial species to their respective hosts. In cell biological terms, many aspects of all the species seem similar but non-identical: the bacteria mostly replicate within epithelial cells; they are taken up by the host cell in an endosome that they customize to generate a cytosolic vacuole; they have to evade cellular defences and have to organize nutrient transport to the vacuole; finally, they have to organize their release to be able to infect the next cell or the next host. What appears to be very difficult and challenging to achieve, is in fact a greatly successful style of parasitism. I will here attempt to cover some of the aspects of the infection biology of Chlamydia, from cell biology to immune defence, epidemiology and possibilities of prevention. I will discuss the pig as a host species and the species known to infect pigs but will in particular draw on the more detailed knowledge that we have on species that infect especially humans. [ABSTRACT FROM AUTHOR]

Intracellular bacteria can multiply inside host cells and manipulate their biology, and the efficacy of traditional antibiotic drug therapy for intracellular bacteria is limited by inadequate drug accumulation. Fighting against these stealthy bacteria has been a longstanding challenge. Here, a system of stimuli-responsive lactoferrin (Lf) nanoparticles is prepared using protein self-assembly technology to deliver broad-spectrum antibiotic rifampicin (Rif) (Rif@Lf NPs) for enhanced infection therapy through targeted elimination of intracellular bacteria. Compared to Rif@BSA NPs, the Rif@Lf NPs can specifically target macrophages infected by bacteria, thus increasing the accumulation of Rif within macrophages. Subsequently, Rif@Lf NPs with positive surface charge further displayed targeted adherence to the bacteria within macrophages and released Rif rapidly in a redoxresponsive manner. Combined with the antibacterial activities of Lf and Rif, the Rif@Lf NPs showed broad-spectrum antibiotic abilities to intracellular bacteria and biofilms. As a result, the Rif@Lf NPs with high safety exhibited excellent therapeutic efficacy in the disease models of subcutaneous infection, sepsis, and bacterial keratitis. Taken together, the antibiotic-loaded Lf nanoparticles present a promising platform to combat pathogen infections through targeted elimination of intracellular bacteria. [ABSTRACT FROM AUTHOR]

Obligate intracellular endobacteria of the genus Wolbachia are widespread in arthropods and several filarial nematodes. Control programs for vector-borne diseases (dengue, Zika, malaria) and anti-filarial therapy with antibiotics are based on this important endosymbiont. Investigating Wolbachia, however, is impeded by the need for host cells. In this study, the requirements for Wolbachia wAlbB growth in a host cell-free in vitro culture system were characterized via qPCRs. A cell lysate fraction from Aedes albopictus C6/36 insect cells containing cell membranes and medium with fetal bovine serum were identified as requisite for cell-free replication of Wolbachia. Supplementation with the membrane fraction of insect cell lysate increased extracellular Wolbachia replication by 4.2-fold. Replication rates in the insect cell-free culture were lower compared to Wolbachia grown inside insect cells. However, the endobacteria were able to replicate for up to 12 days and to infect uninfected C6/36 cells. Cellfree Wolbachia treated with the lipid II biosynthesis inhibitor fosfomycin had an enlarged phenotype, seen previously for intracellular Wolbachia in C6/36 cells, indicating that the bacteria were unable to divide. In conclusion, we have developed a cell-free culture system in which Wolbachia replicate for up to 12 days, providing an in vitro tool to elucidate the biology of these endobacteria, e.g., cell division by using compounds that may not enter the C6/36 cells. A better understanding of Wolbachia biology, and in particular host-symbiont interactions, is key to the use of Wolbachia in vector control programs and to future drug development against filarial diseases. [ABSTRACT FROM AUTHOR]

Infections caused by intracellular bacterial pathogens are difficult to treat since most antibiotics have low cell permeability and undergo rapid degradation within cells. The rapid development and dissemination of antimicrobial–resistant strains have exacerbated this dilemma. With the increasing knowledge of host–pathogen interactions, especially bacterial strategies for survival and proliferation within host cells, host-directed therapy (HDT) has attracted increased interest and has emerged as a promising anti-infection method for treating intracellular infection. Herein, we applied a cell-based screening approach to a US Food and Drug Administration (FDA)-approved drug library to identify compounds that can inhibit the intracellular replication of Salmonella Typhimurium (S. Typhimurium). This screening allowed us to identify the antidiarrheal agent loperamide (LPD) as a potent inhibitor of S. Typhimurium intracellular proliferation. LPD treatment of infected cells markedly promoted the host autophagic response and lysosomal activity. A mechanistic study revealed that the increase in host autophagy and elimination of intracellular bacteria were dependent on the high expression of glycoprotein nonmetastatic melanoma protein B (GPNMB) induced by LPD. In addition, LPD treatment effectively protected against S. Typhimurium infection in Galleria mellonella and mouse models. Thus, our study suggested that LPD may be useful for the treatment of diseases caused by intracellular bacterial pathogens. Moreover, LPD may serve as a promising lead compound for the development of anti-infection drugs based on the HDT strategy.

The International Committee on Systematics of Prokaryotes (ICSP) discussed and rejected in 2020 a proposal to modify the International Code of Nomenclature of Prokaryotes to allow the use of gene sequences as type for naming prokaryotes. An alternative nomenclatural code, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), which considers genome sequences as type material for naming species, was published in 2022. Members of the ICSP subcommittee for the taxonomy of the phylum Chlamydiae (Chlamydiota) consider that the use of gene sequences as type would benefit the taxonomy of microorganisms that are difficult to culture such as the chlamydiae and other strictly intracellular bacteria. We recommend the registration of new names of uncultured prokaryotes in the SeqCode registry.

Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8+ T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8+ T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12–200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections. [Display omitted] [ABSTRACT FROM AUTHOR]

Chlamydial infections and diseases caused by filarial nematodes are global health concerns. However, treatment presents challenges due to treatment failures potentially caused by persisting Chlamydia and long regimens against filarial infections accompanied by low compliance. A new treatment strategy could be the targeting of the reduced peptidoglycan structures involved in cell division in the obligate intracellular bacteria Chlamydia and Wolbachia, the latter being obligate endosymbionts supporting filarial development, growth, and survival. Here, cell culture experiments with C. trachomatis and Wolbachia showed that the nucleoside antibiotics muraymycin and carbacaprazamycin interfere with bacterial cell division and induce enlarged, aberrant cells resembling the penicillin-induced persistence phenotype in Chlamydia. Enzymatic inhibition experiments with purified C. pneumoniae MraY revealed that muraymycin derivatives abolish the synthesis of the peptidoglycan precursor lipid I. Comparative in silico analyses of chlamydial and wolbachial MraY with the corresponding well-characterized enzyme in Aquifex aeolicus revealed a high degree of conservation, providing evidence for a similar mode of inhibition. Muraymycin D2 treatment eradicated persisting non-dividing C. trachomatis cells from an established penicillin-induced persistent infection. This finding indicates that nucleoside antibiotics may have additional properties that can break bacterial persistence. [ABSTRACT FROM AUTHOR]

Chlamydiae are bacteria that are intriguing and important at the same time. The genus Chlamydia encompasses many species of obligate intracellular organisms: they can multiply only inside the cells of their host organism. Many, perhaps most animals have their own specifically adapted chlamydial species. In humans, the clinically most relevant species is Chlamydia trachomatis, which has particular importance as an agent of sexually transmitted disease. Pigs are the natural host of Chlamydia suis but may also carry Chlamydia abortus and Chlamydia pecorum. C. abortus and possibly C. suis have anthropozoonotic potential, which makes them interesting to human medicine, but all three species bring a substantial burden of disease to pigs. The recent availability of genomic sequence comparisons suggests adaptation of chlamydial species to their respective hosts. In cell biological terms, many aspects of all the species seem similar but non-identical: the bacteria mostly replicate within epithelial cells; they are taken up by the host cell in an endosome that they customize to generate a cytosolic vacuole; they have to evade cellular defences and have to organize nutrient transport to the vacuole; finally, they have to organize their release to be able to infect the next cell or the next host. What appears to be very difficult and challenging to achieve, is in fact a greatly successful style of parasitism. I will here attempt to cover some of the aspects of the infection biology of Chlamydia, from cell biology to immune defence, epidemiology and possibilities of prevention. I will discuss the pig as a host species and the species known to infect pigs but will in particular draw on the more detailed knowledge that we have on species that infect especially humans.

Within epithelial cells, Pseudomonas aeruginosa depends on its type III secretion system (T3SS) to escape vacuoles and replicate rapidly in the cytosol. Previously, it was assumed that intracellular subpopulations remaining T3SS-negative (and therefore in vacuoles) were destined for degradation in lysosomes, supported by data showing vacuole acidification. Here, we report in both corneal and bronchial human epithelial cells that vacuole-associated bacteria can persist, sometimes in the same cells as cytosolic bacteria. Using a combination of phase-contrast, confocal, and correlative light-electron microscopy (CLEM), we also found they can demonstrate biofilm-associated markers: cdrA and cyclic-di-GMP (c-di-GMP). Vacuolar-associated bacteria, but not their cytosolic counterparts, tolerated the cell-permeable antibiotic ofloxacin. Surprisingly, use of mutants showed that both persistence in vacuoles and ofloxacin tolerance were independent of the biofilm-associated protein CdrA or exopolysaccharides (Psl, Pel, alginate). A T3SS mutant (ΔexsA) unable to escape vacuoles phenocopied vacuole-associated subpopulations in wild-type PAO1-infected cells, with results revealing that epithelial cell death depended upon bacterial viability. Intravital confocal imaging of infected mouse corneas confirmed that P. aeruginosa formed similar intracellular subpopulations within epithelial cells in vivo. Together, these results show that P. aeruginosa differs from other pathogens by diversifying intracellularly into vacuolar and cytosolic subpopulations that both contribute to pathogenesis. Their different gene expression and behavior (e.g., rapid replication versus slow replication/persistence) suggest cooperation favoring both short- and long-term interests and another potential pathway to treatment failure. How this intracellular diversification relates to previously described "acute versus chronic" virulence gene-expression phenotypes of P. aeruginosa remains to be determined. IMPORTANCE Pseudomonas aeruginosa can cause sight- and life-threatening opportunistic infections, and its evolving antibiotic resistance is a growing concern. Most P. aeruginosa strains can invade host cells, presenting a challenge to therapies that do not penetrate host cell membranes. Previously, we showed that the P. aeruginosa type III secretion system (T3SS) plays a pivotal role in survival within epithelial cells, allowing escape from vacuoles, rapid replication in the cytoplasm, and suppression of host cell death. Here, we report the discovery of a novel T3SS-negative subpopulation of intracellular P. aeruginosa within epithelial cells that persist in vacuoles rather than the cytoplasm and that tolerate a cell-permeable antibiotic (ofloxacin) that is able to kill cytosolic bacteria. Classical biofilm-associated markers, although demonstrated by this subpopulation, are not required for vacuolar persistence or antibiotic tolerance. These findings advance our understanding of how P. aeruginosa hijacks host cells, showing that it diversifies into multiple populations with T3SS-negative members enabling persistence while rapid replication is accomplished by more vulnerable T3SS-positive siblings. Intracellular P. aeruginosa persisting and tolerating antibiotics independently of the T3SS or biofilm-associated factors could present additional challenges to development of more effective therapeutics.

Phytoplasmas are intracellular pathogenic bacteria that infect a wide range of plant species, including agriculturally important crops and ornamental trees. However, our understanding of the relationship between symptom severity, disease progression, and phytoplasma concentration remains limited due to the inability to inoculate phytoplasmas mechanically into new plant hosts. The present study investigated phytoplasma titer dynamics and symptom development in periwinkle and tomato, both infected with the same potato purple top (PPT) phytoplasma strain using a small seedling grafting approach. Virescence, phyllody, and witches'-broom (WB) symptoms sequentially developed in periwinkle, while in tomato plants, big bud (BB, a form of phyllody), cauliflower-like inflorescence (CLI), and WB appeared in order. Results from quantitative polymerase chain reaction (qPCR) targeting the PPT phytoplasma's 16S rRNA gene revealed that in both host species, phytoplasma titers differed significantly at different infection stages. Notably, the highest phytoplasma concentration in periwinkles was observed in samples displaying phyllody symptoms, whereas in tomatoes, the titer peaked at the BB stage. Western blot analysis, utilizing an antibody specific to PPT phytoplasma, confirmed substantial phytoplasma presence in samples displaying phyllody and BB symptoms, consistent with the qPCR results. These findings challenge the conventional understanding that phytoplasma infection dynamics result in a higher titer at later stages, such as WB (excessive vegetative growth), rather than in the early stage, such as phyllody (abnormal reproductive growth). Furthermore, the PPT phytoplasma titer was markedly higher in periwinkles than in tomato plants, indicating differing susceptibilities between the hosts. This study reveals distinct host responses to PPT phytoplasma infection, providing valuable insights into phytoplasma titer dynamics and symptom development, with implications for the future management of agricultural disease. [ABSTRACT FROM AUTHOR]

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as ß-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche. [ABSTRACT FROM AUTHOR]

Obligate intracellular endobacteria of the genus Wolbachia are widespread in arthropods and several filarial nematodes. Control programs for vector-borne diseases (dengue, Zika, malaria) and anti-filarial therapy with antibiotics are based on this important endosymbiont. Investigating Wolbachia, however, is impeded by the need for host cells. In this study, the requirements for Wolbachia wAlbB growth in a host cell-free in vitro culture system were characterized via qPCRs. A cell lysate fraction from Aedes albopictus C6/36 insect cells containing cell membranes and medium with fetal bovine serum were identified as requisite for cell-free replication of Wolbachia. Supplementation with the membrane fraction of insect cell lysate increased extracellular Wolbachia replication by 4.2-fold. Replication rates in the insect cell-free culture were lower compared to Wolbachia grown inside insect cells. However, the endobacteria were able to replicate for up to 12 days and to infect uninfected C6/36 cells. Cell-free Wolbachia treated with the lipid II biosynthesis inhibitor fosfomycin had an enlarged phenotype, seen previously for intracellular Wolbachia in C6/36 cells, indicating that the bacteria were unable to divide. In conclusion, we have developed a cell-free culture system in which Wolbachia replicate for up to 12 days, providing an in vitro tool to elucidate the biology of these endobacteria, e.g., cell division by using compounds that may not enter the C6/36 cells. A better understanding of Wolbachia biology, and in particular host-symbiont interactions, is key to the use of Wolbachia in vector control programs and to future drug development against filarial diseases.

Chlamydia pneumoniae infection cases have usually accounted for

Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN‐7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella‐containing vacuoles after long‐term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread. [ABSTRACT FROM AUTHOR]

Simple Summary: Bacterial infections have traditionally been identified by culture; however, with advances in the understanding of the role of bacteria in disease, it is recognized that intracellular bacteria can be visualized within cells. Fluorescent in situ hybridization (FISH) is a technique used to not only identify intracellular organisms but also allows researchers to see them, in situ. In this review, we discuss the use of FISH for the identification of intracellular bacteria in companion animals. Most studies have focused on the identification of Escherichia coli, particularly within the gastrointestinal tract. Additionally, bacterial associations with inflammatory diseases affecting the liver, kidney, skin, and cardiovascular systems have been investigated with mixed results. FISH techniques have been applied for the visualization and identification of intracellular bacteria in companion animal species. Most frequently, these techniques have focused on the identification of adhesive-invasive Escherichia coli in gastrointestinal disease, although various other organisms have been identified in inflammatory or neoplastic gastrointestinal disease. Previous studies have investigated a potential role of Helicobacter spp. in inflammatory gastrointestinal and hepatic conditions. Other studies evaluating the role of infectious organisms in hepatopathies have received some attention with mixed results. FISH techniques using both eubacterial and species-specific probes have been applied in inflammatory cardiovascular, urinary, and cutaneous diseases to screen for intracellular bacteria. This review summarizes the results of these studies. [ABSTRACT FROM AUTHOR]

Chemo‐immunotherapy has shown great success in boosting systemic anti‐tumor effects in the clinic. However, cancer cells can escape the maximum impact of chemotherapy through intracellular symbiotic bacteria and abnormally activated macropinocytosis, while cancer immune evasion is largely facilitated by the programmed cell death 1 ligand 1 (PD‐L1) protein and tumor‐secreted exosomes. To efficiently sensitize chemo‐immunotherapy, the clinical mitoxantrone (MTO) is therefore rationally coordinated with Cu2+ to develop nanoscale metal‐organic frameworks (MTO‐Cu), then loaded with the macropinocytosis and exosome secretion inhibitor amiloride (AMI) and modified with targeted chondroitin sulfate (CS) to form CS/MTO‐Cu@AMI nanoadjuvant. Notably, the coordinated Cu2+ effectively triggers cuproptosis‐induced mitochondrial dysfunction, which activates AMPK pathway‐mediated PD‐L1 protein degradation, deprives energy supply for macropinocytosis and exosome release, and amplifies oxidative stress for deactivating intracellular bacteria, thus efficiently sensitizing chemo‐immunotherapy. Meanwhile, AMI suppresses macropinocytosis and exosome secretion to act synergistically with Cu2+‐caused chemo‐immunotherapy potentiation. Moreover, damaged dsDNA during CS/MTO‐Cu@AMI treatment activates the cGAS‐STING pathway, further evoking the anti‐tumor immunity. Additionally, the attached CS endows CS/MTO‐Cu@AMI with specific tumor targeting and hyaluronidase‐responsive charge reversal property, while MTO‐Cu endows this nanoadjuvant with pH/GSH dual‐responsive release behavior. Therefore, CS/MTO‐Cu@AMI efficiently sensitizes chemotherapy and activates systemic antitumor immunity in vitro and in vivo, providing an innovative solution to potentiate cancer chemo‐immunotherapy. [ABSTRACT FROM AUTHOR]

Breast cancer (BC) is a prevalent malignancy. There exists a strong association between gut microbiota (GM) and the development of BC. The GM composition in individuals with BC significantly differs from that in their healthy counterparts. Furthermore, the distribution of GM varies significantly among individuals with different types of BC. The GM can impact BC through metabolite secretion, the gut-mammary axis, and other pathways. Modulating the GM can serve as a very promising potential therapeutic strategy in the treatment of BC. This article will summarize existing research, focusing on the relationship between intestinal microbiota and BC. At the same time, the project will also analyze the application value of intestinal microorganisms in BC intervention work, so as to provide a reference for the further development of BC prevention and treatment work. [ABSTRACT FROM AUTHOR]

ABSTRACTBrucella is a zoonotic intracellular bacterium that poses threats to human health and economic security. Intracellular infection is a hallmark of the agent Brucella and a primary cause of distress, through which the bacterium regulates the host intracellular environment to promote its own colonization and replication, evading host immunity and pharmaceutical killing. Current studies of Brucella intracellular processes are typically premised on bacterial phenotype such as intracellular bacterial survival, followed by biochemical or molecular biological approaches to reveal detailed mechanisms. While such processes can deepen the understanding of Brucella-host interaction, the insights into host alterations in infection would be easily restricted to known pathways. In the current study, we applied CRISPR Cas9 screen to identify host genes that are most affected by Brucella infection on cell viability at the genomic level. As a result of CRISPR screening, we firstly identified that knockout of the negatively selected genes GOLGA6L6, DEFB103B, OR4F29, and ERCC6 attenuate the viability of both the host cells and intracellular Brucella, suggesting these genes to be potential therapeutic targets for Brucella control. In particular, knockout of DEFB103B diminished Brucella intracellular survival by altering host cell autophagy. Conversely, knockout of positive screening genes promoted intracellular proliferation of Brucella. In summary, we screened host genes at the genomic level throughout Brucella infection, identified host genes that are previously not recognized to be involved in Brucella infection, and provided targets for intracellular infection control.IMPORTANCEBrucella is a Gram-negative bacterium that infects common mammals causing arthritis, myalgia, neuritis, orchitis, or miscarriage and is difficult to cure with antibiotics due to its intracellular parasitism. Therefore, unraveling the mechanism of Brucella-host interactions will help controlling Brucella infections. CRISPR-Cas9 is a gene editing technology that directs knockout of individual target genes by guided RNA, from which genome-wide gene-knockout cell libraries can be constructed. Upon infection with Brucella, the cell library would show differences in viability as a result of the knockout and specific genes could be revealed by genomic DNA sequencing. As a result, genes affecting cell viability during Brucella infection were identified. Further testing of gene function may reveal the mechanisms of Brucella-host interactions, thereby contributing to clinical therapy.

Abstract Introduction Tumor‐associated bacteria and gut microbiota have gained significant attention in recent years due to their potential role in cancer development and therapeutic response. This review aims to discuss the contributions of intratumor bacteria outside the gastrointestinal tract, in addition to exploring the mechanisms, functions, and implications of these bacteria in cancer therapy. Methods We reviewed current literature on intratumor bacteria and their impact on tumorigenesis, progression, metastasis, drug resistance, and anti‐tumor immune modulation. Additionally, we examined techniques used to detect intratumor bacteria, precautions necessary when handling low microbial biomass tumor samples, and the recent progress in bacterial manipulation for tumor treatment. Results Research indicates that each type of cancer uniquely interacts with its microbiome, and bacteria can be detected even in non‐gastrointestinal tumors with low bacterial abundance. Intracellular bacteria have the potential to regulate tumor cells' biological behavior and contribute to critical aspects of tumor development. Furthermore, bacterial‐based anti‐tumor therapies have shown promising results in cancer treatment. Conclusions Understanding the complex interactions between intratumor bacteria and tumor cells could lead to the development of more precise cancer treatment strategies. Further research into non‐gastrointestinal tumor‐associated bacteria is needed to identify new therapeutic approaches and expand our knowledge of the microbiota's role in cancer biology.

Chlamydial infections and diseases caused by filarial nematodes are global health concerns. However, treatment presents challenges due to treatment failures potentially caused by persisting Chlamydia and long regimens against filarial infections accompanied by low compliance. A new treatment strategy could be the targeting of the reduced peptidoglycan structures involved in cell division in the obligate intracellular bacteria Chlamydia and Wolbachia, the latter being obligate endosymbionts supporting filarial development, growth, and survival. Here, cell culture experiments with C. trachomatis and Wolbachia showed that the nucleoside antibiotics muraymycin and carbacaprazamycin interfere with bacterial cell division and induce enlarged, aberrant cells resembling the penicillin-induced persistence phenotype in Chlamydia. Enzymatic inhibition experiments with purified C. pneumoniae MraY revealed that muraymycin derivatives abolish the synthesis of the peptidoglycan precursor lipid I. Comparative in silico analyses of chlamydial and wolbachial MraY with the corresponding well-characterized enzyme in Aquifex aeolicus revealed a high degree of conservation, providing evidence for a similar mode of inhibition. Muraymycin D2 treatment eradicated persisting non-dividing C. trachomatis cells from an established penicillin-induced persistent infection. This finding indicates that nucleoside antibiotics may have additional properties that can break bacterial persistence.

Seed-feeding Nysius insects (Hemiptera: Lygaeidae) have a symbiotic association with distinct intracellular bacteria, "Candidatus Schneideria nysicola" (Gammaproteobacteria). Although many other hemipteran insect groups generally rely on bacterial symbionts that synthesize all ten essential amino acids lacking in their plant sap diets, the nutritional role of Schneideria in Nysius hosts that specialize on a more nutritionally complete seed-based diet has remained unknown. To determine the nutritional and functional capabilities of Schneideria, we sequenced the complete Schneideria genomes from three distantly related endemic Hawaiian Nysius seed bug species. The complete Schneideria genomes are highly conserved and perfectly syntenic among Hawaiian Nysius host species. Each circular chromosome is ∼0.57 Mb in size and encodes 537 protein-coding genes. They further exhibit a strong A + T nucleotide substitution bias with an average G + C nucleotide content of 29%. The predicted nutritional contribution of Schneideria includes four B vitamins and five of the ten essential amino acids that likely match its hosts' seed-based diet. Disrupted and degraded genes in Schneideria suggests that Hawaiian lineages are undergoing continued gene losses observed in the smaller genomes of the other more ancient hemipteran symbionts.

ABSTRACTMycobacterium abscessus is a nontuberculous mycobacteria that contributes to the decline and death of patients with lung diseases such as cystic fibrosis and other muco-obstructive airway diseases. M. abscessus is challenging to treat due to its extensive antibiotic resistance and ability to survive inside mammalian cells. An alternative to antibiotics is the therapeutic use of bacteriophages (phages). There are recent cases of phage therapy being used to treat M. abscessus infections in people under compassionate-use conditions. However, little is known about the ability of phages to kill bacteria, such as M. abscessus, which reside in an intracellular environment. Here, we used M. abscessus strains and phages from recent phage therapy cases to determine if phages can enter mammalian cells and if they can infect and kill intracellular M. abscessus. Using fluorescence microscopy, we demonstrate phage uptake by macrophages and lung epithelial cells, and we further demonstrate phage infection of intracellular M. abscessus with fluorescent reporter phages. Transmission electron microscopy was additionally used to image phage infection of intracellular M. abscessus. Together, these findings provide the first visualizations of phage-M. abscessus interactions in an intracellular environment. Finally, we show that phage treatment can significantly reduce the intracellular burden of M. abscessus in a manner that depends on both the specific phage and mammalian cell type involved. These results demonstrate the potential to use phage therapy to treat intracellular bacteria, specifically M. abscessus, while also highlighting the importance of prescreening phage therapy candidates for activity in an intracellular environment.IMPORTANCEAs we rapidly approach a post-antibiotic era, bacteriophage (phage) therapy may offer a solution for treating drug-resistant bacteria. Mycobacterium abscessus is an emerging, multidrug-resistant pathogen that causes disease in people with cystic fibrosis, chronic obstructive pulmonary disease, and other underlying lung diseases. M. abscessus can survive inside host cells, a niche that can limit access to antibiotics. As current treatment options for M. abscessus infections often fail, there is an urgent need for alternative therapies. Phage therapy is being used to treat M. abscessus infections as an option of last resort. However, little is known about the ability of phages to kill bacteria in the host environment and specifically in an intracellular environment. Here, we demonstrate the ability of phages to enter mammalian cells and to infect and kill intracellular M. abscessus. These findings support the use of phages to treat intracellular bacterial pathogens.

ABSTRACT Pathogenic Rickettsia species proliferate within the cytoplasm of permissive host cells in vivo. The cytoplasm of these host cells is adequate to support the complex metabolic and physiological needs for Rickettsia growth. However, a dramatic host/pathogen interplay occurs when Rickettsia encounter innate immune cells, whereby the bacteria can proliferate as normal or the host can restrict bacterial growth. This interplay is most divergent within myeloid host cells, where intra- and extracellular factors can produce either successful Rickettsia parasitism or innate immune control of bacterial proliferation. With the prior knowledge that the mammalian complement system is activated during mammalian infection, we sought to determine if extracellular complement activation and anaphylatoxin signaling can modify the fate of Rickettsia within mononuclear host cells. Results indicate that supplementation of growth media with either C3a or C5a anaphylatoxin peptides is sufficient for many myeloid cells to control the proliferation of multiple different Rickettsia species. Chemical or genetic disruption of anaphylatoxin signaling or anaphylatoxin receptors eliminates complement-induced restriction of bacterial proliferation. Finally, anaphylatoxin signaling modifies macrophage physiology by inducing inflammatory phenotypes that ultimately control the intracellular proliferation of these pathogens. IMPORTANCE Pathogenic Rickettsia species are extremely dangerous bacteria that grow within the cytoplasm of host mammalian cells. In most cases, these bacteria are able to overpower the host cell and grow within the protected environment of the cytoplasm. However, a dramatic conflict occurs when Rickettsia encounter innate immune cells; the bacteria can “win” by taking over the host, or the bacteria can “lose” if the host cell efficiently fights the infection. This manuscript examines how the immune complement system is able to detect the presence of Rickettsia and alert nearby cells. Byproducts of complement activation called anaphylatoxins are signals that “activate” innate immune cells to mount an aggressive defensive strategy. This study enhances our collective understanding of the innate immune reaction to intracellular bacteria and will contribute to future efforts at controlling these dangerous infections.

Breast cancer can harbor intracellular bacteria, which may have an impact on metastasis and therapeutic responses. Silver nanoparticles are FDA-approved for their antimicrobial potential, plus they have pleiotropic benefits for eradicating cancer cells. In the current work we synthesized photothermal silver nanoparticles (AgNPs) with an absorption at 800 nm for heat generation when exposed to near-infrared laser irradiation. Breast cell lines MCF 10A, MCF7, and MDA MB 231 were infected with Pseudomonas aeruginosa, and their response to AgNPs, heat, or photothermal therapy (PTT) was evaluated. The results demonstrate that the application of a brief heating of cells treated with AgNPs offers a synergistic benefit in killing both infected and non-infected cells. Using 10 µg/mL of AgNPs plus laser stimulation induced a temperature change of 12 °C, which was sufficient for reducing non-infected breast cells by 81–94%. Infected breast cells were resistant to PTT, with only a reduction of 45–68%. In the absence of laser stimulation, 10 µg/mL of AgNPs reduced breast cell populations by 10–65% with 24 h of exposure. This concentration had no impact on the survival of planktonic bacteria with or without laser stimulation, although infected breast cells had a 42–90% reduction in intracellular bacteria. Overall, this work highlights the advantages of AgNPs for the generation of heat, and to augment the benefits of heat, in breast cancer cells harboring intracellular infection. [ABSTRACT FROM AUTHOR]

Ticks are vectors of numerous agents of medical importance and may be infected by various Chlamydia-related bacteria, such as members of Parachlamydiaceae and Rhabdochlamydiaceae families, which are sharing the same biphasic life cycle with the pathogenic Chlamydia. However, the veterinary importance of ticks and of their internalized pathogens remains poorly studied. Thus, we wondered (i) whether the prevalence of ticks was higher in zoological gardens than in control areas with similar altitude, vegetation, humidity and temperature, and (ii) whether the presence of Chlamydia-related bacteria in ticks may vary according to the environment in which the ticks are collected. A total of 212 Ixodes ricinus ticks were collected, and all were tested for the presence of DNA from any member of the Chlamydiae phylum using a pan-Chlamydiae quantitative PCR (qPCR). We observed a higher prevalence of ticks outside animal enclosures in both zoos, compared to in enclosures. Tick prevalence was also higher outside zoos, compared to in enclosures. With 30% (3/10) of infected ticks, the zoological gardens presented a prevalence of infected ticks that was higher than that in contiguous areas (13.15%, 10/76), and higher than the control distant areas (8.65%, 9/104). In conclusion, zoological gardens in Switzerland appear to contain fewer ticks than areas outside zoological gardens. However, ticks from zoos more often contain Chlamydia-like organisms than ticks from contiguous or distant control areas. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis infection of ocular conjunctiva can lead to blindness, while infection of the female genital tract can lead to chronic pelvic pain, ectopic pregnancy, and/or infertility. Conjunctival and fallopian tube inflammation and the resulting disease sequelae are attributed to immune responses induced by chlamydial infection at these mucosal sites. The conserved chlamydial plasmid has been implicated in enhancing infection, via improved host cell entry and exit, and accelerating innate inflammatory responses that lead to tissue damage. The chlamydial plasmid encodes eight open reading frames, three of which have been associated with virulence: a secreted protein, Pgp3, and putative transcriptional regulators, Pgp4 and Pgp5. Although Pgp3 is an important plasmid-encoded virulence factor, recent studies suggest that chlamydial plasmid-mediated virulence extends beyond the expression of Pgp3. In this review, we discuss studies of genital, ocular, and gastrointestinal infection with C. trachomatis or C. muridarum that shed light on the role of the plasmid in disease development, and the potential for tissue and species-specific differences in plasmid-mediated pathogenesis. We also review evidence that plasmidassociated inflammation can be independent of bacterial burden. The functions of each of the plasmid-encoded proteins and potential molecular mechanisms for their role(s) in chlamydial virulence are discussed. Although the understanding of plasmid-associated virulence has expanded within the last decade, many questions related to how and to what extent the plasmid influences chlamydial infectivity and inflammation remain unknown, particularly with respect to human infections. Elucidating the answers to these questions could improve our understanding of how chlamydia augment infection and inflammation to cause disease. [ABSTRACT FROM AUTHOR]

Introduction: Tumor‐associated bacteria and gut microbiota have gained significant attention in recent years due to their potential role in cancer development and therapeutic response. This review aims to discuss the contributions of intratumor bacteria outside the gastrointestinal tract, in addition to exploring the mechanisms, functions, and implications of these bacteria in cancer therapy. Methods: We reviewed current literature on intratumor bacteria and their impact on tumorigenesis, progression, metastasis, drug resistance, and anti‐tumor immune modulation. Additionally, we examined techniques used to detect intratumor bacteria, precautions necessary when handling low microbial biomass tumor samples, and the recent progress in bacterial manipulation for tumor treatment. Results: Research indicates that each type of cancer uniquely interacts with its microbiome, and bacteria can be detected even in non‐gastrointestinal tumors with low bacterial abundance. Intracellular bacteria have the potential to regulate tumor cells' biological behavior and contribute to critical aspects of tumor development. Furthermore, bacterial‐based anti‐tumor therapies have shown promising results in cancer treatment. Conclusions: Understanding the complex interactions between intratumor bacteria and tumor cells could lead to the development of more precise cancer treatment strategies. Further research into non‐gastrointestinal tumor‐associated bacteria is needed to identify new therapeutic approaches and expand our knowledge of the microbiota's role in cancer biology. [ABSTRACT FROM AUTHOR]

Identifying suitable animal models and standardizing preclinical methods are important for the generation, characterization, and development of new vaccines, including those against Francisella tularensis. Non-human primates represent an important animal model to evaluate tularemia vaccine efficacy, and the use of correlates of vaccine-induced protection may facilitate bridging immune responses from non-human primates to people. However, among small animals, Fischer 344 rats represent a valuable resource for initial studies to evaluate immune responses, to identify correlates of protection, and to screen novel vaccines. In this study, we performed a comparative analysis of three Fischer rat substrains to determine potential differences in immune responses, to evaluate methods used to quantify potential correlates of protection, and to evaluate protection after vaccination. To this end, we took advantage of data previously generated using one of the rat substrains by evaluating two live vaccines, LVS and F. tularensis SchuS4-ΔclpB (ΔclpB). We compared immune responses after primary vaccination, adaptive immune responses upon re-stimulation of leukocytes in vitro, and sensitivity to aerosol challenge. Despite some detectable differences, the results highlight the similarity of immune responses to tularemia vaccines and challenge outcomes between the three substrains, indicating that all offer acceptable and comparable approaches as animal models to study Francisella infection and immunity. [ABSTRACT FROM AUTHOR]

The International Committee on Systematics of Prokaryotes (ICSP) discussed and rejected in 2020 a proposal to modify the International Code of Nomenclature of Prokaryotes to allow the use of gene sequences as type for naming prokaryotes. An alternative nomenclatural code, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), which considers genome sequences as type material for naming species, was published in 2022. Members of the ICSP subcommittee for the taxonomy of the phylum Chlamydiae (Chlamydiota) consider that the use of gene sequences as type would benefit the taxonomy of microorganisms that are difficult to culture such as the chlamydiae and other strictly intracellular bacteria. We recommend the registration of new names of uncultured prokaryotes in the SeqCode registry.

ABSTRACT The obligate intracellular bacterium Chlamydia trachomatis inserts a family of inclusion membrane (Inc) proteins into the membrane of its vacuole (the inclusion). The Inc CpoS is a critical suppressor of host cellular immune surveillance, but the underlying mechanism remained elusive. By complementing a cpoS mutant with various natural orthologs and variants of CpoS, we linked distinct molecular interactions of CpoS to distinct functions. Unexpectedly, we found CpoS to be essential for the formation of inclusion membrane microdomains that control the spatial organization of multiple Incs involved in signaling and modulation of the host cellular cytoskeleton. While the function of CpoS in microdomains was uncoupled from its role in the suppression of host cellular defenses, we found the ability of CpoS to interact with Rab GTPases to be required not only for the manipulation of membrane trafficking, such as to mediate transport of ceramide-derived lipids (sphingolipids) to the inclusion, but also for the inhibition of Stimulator of interferon genes (STING)-dependent type I interferon responses. Indeed, depletion of Rab35 phenocopied the exacerbated interferon responses observed during infection with CpoS-deficient mutants. Overall, our findings highlight the role of Inc–Inc interactions in shaping the inclusion microenvironment and the modulation of membrane trafficking as a pathogenic immune evasion strategy. IMPORTANCE Chlamydia trachomatis is a prevalent bacterial pathogen that causes blinding ocular scarring and urogenital infections that can lead to infertility and pregnancy complications. Because Chlamydia can only grow within its host cell, boosting the intrinsic defenses of human cells may represent a novel strategy to fight pathogen replication and survival. Hence, CpoS, a Chlamydia protein known to block host cellular defenses, or processes regulated by CpoS, could provide new opportunities for therapeutic intervention. By revealing CpoS as a multifunctional virulence factor and by linking its ability to block host cellular immune signaling to the modulation of membrane trafficking, the present work may provide a foundation for such rationale targeting and advances our understanding of how intracellular bacteria can shape and protect their growth niche.

Chlamydia trachomatis infection of ocular conjunctiva can lead to blindness, while infection of the female genital tract can lead to chronic pelvic pain, ectopic pregnancy, and/or infertility. Conjunctival and fallopian tube inflammation and the resulting disease sequelae are attributed to immune responses induced by chlamydial infection at these mucosal sites. The conserved chlamydial plasmid has been implicated in enhancing infection, via improved host cell entry and exit, and accelerating innate inflammatory responses that lead to tissue damage. The chlamydial plasmid encodes eight open reading frames, three of which have been associated with virulence: a secreted protein, Pgp3, and putative transcriptional regulators, Pgp4 and Pgp5. Although Pgp3 is an important plasmid-encoded virulence factor, recent studies suggest that chlamydial plasmid-mediated virulence extends beyond the expression of Pgp3. In this review, we discuss studies of genital, ocular, and gastrointestinal infection with C. trachomatis or C. muridarum that shed light on the role of the plasmid in disease development, and the potential for tissue and species-specific differences in plasmid-mediated pathogenesis. We also review evidence that plasmid-associated inflammation can be independent of bacterial burden. The functions of each of the plasmid-encoded proteins and potential molecular mechanisms for their role(s) in chlamydial virulence are discussed. Although the understanding of plasmid-associated virulence has expanded within the last decade, many questions related to how and to what extent the plasmid influences chlamydial infectivity and inflammation remain unknown, particularly with respect to human infections. Elucidating the answers to these questions could improve our understanding of how chlamydia augment infection and inflammation to cause disease.

Introduction: Tularemia is mainly caused by Francisella tularensis (Ft) subsp. tularensis (Ftt) and Ft subsp. holarctica (Ftt) in humans and in more than 200 animal species including rabbits and hares. Human clinical manifestations depend on the route of infection and range from flu-like symptoms to severe pneumonia with a mortality rate up to 60% without treatment. So far, only 2D cell culture and animal models are used to study Francisella virulence, but the gained results are transferable to human infections only to a certain extent. Method: In this study, we firstly established an ex vivo human lung tissue infection model using different Francisella strains: Ftt Life Vaccine Strain (LVS), Ftt LVS ΔiglC, Ftt human clinical isolate A-660 and a German environmental Francisella species strain W12-1067 (F-W12). Human lung tissue was used to determine the colony forming units and to detect infected cell types by using spectral immunofluorescence and electron microscopy. Chemokine and cytokine levels were measured in culture supernatants. Results: Only LVS and A-660 were able to grow within the human lung explants, whereas LVS ΔiglC and F-W12 did not replicate. Using human lung tissue, we observed a greater increase of bacterial load per explant for patient isolate A-660 compared to LVS, whereas a similar replication of both strains was observed in cell culture models with human macrophages. Alveolar macrophages were mainly infected in human lung tissue, but Ftt was also sporadically detected within white blood cells. Although Ftt replicated within lung tissue, an overall low induction of pro-inflammatory cytokines and chemokines was observed. A-660-infected lung explants secreted slightly less of IL-1β, MCP-1, IP-10 and IL-6 compared to Ftt LVS-infected explants, suggesting a more repressed immune response for patient isolate A-660. When LVS and A-660 were used for simultaneous co-infections, only the ex vivo model reflected the less virulent phenotype of LVS, as it was outcompeted by A-660. Conclusion: We successfully implemented an ex vivo infection model using human lung tissue for Francisella. The model delivers considerable advantages and is able to discriminate virulent Francisella from less-or non-virulent strains and can be used to investigate the role of specific virulence factors. [ABSTRACT FROM AUTHOR]

Cancer metastasis is a crucial step of malignancy progression determined by cancer cell-intrinsic properties and extrinsic environmental factors. The intratumor microbiota is a novel cancer component that functionally impinges on tumor progression in broad cancer types. The intratumor microbiota is present in tumor tissue at low abundance and low diversity, with predominant intracellular localization. The intratumor microbiota is implicated in distinguishing normal versus cancer tissue, drug responder versus nonresponder cancer, good versus bad prognosis, and metastatic versus non-metastatic cancers. Modulation of the intratumor microbiota can reduce cancer metastasis, impede cancer progression, and reprogram the immune response. Cancer metastasis is the leading cause of mortality in patients with cancer. Theories have been developed to explain the causes and principles of metastasis. Metastasis is attributed to cancer cell-intrinsic properties and the extrinsic cellular environment. In recent years, the intratumor microbiota has been identified as an integral tumor component and may functionally regulate various aspects of metastasis. These novel discoveries in intratumor microbiota reshape the framework of our understanding of metastasis and reveal a new path for studies on cancer progression and clinical cancer management. Here, we summarize recent advances in the emerging roles of intratumor microbiota in cancer metastasis and discuss the challenges and implications for cancer treatment. [ABSTRACT FROM AUTHOR]

Osteomyelitis is an infection of the bone that is often difficult to treat and causes a significant healthcare burden. Staphylococcus aureus is the most common pathogen causing osteomyelitis. Osteomyelitis mouse models have been established to gain further insights into the pathogenesis and host response. Here, we use an established S. aureus hematogenous osteomyelitis mouse model to investigate morphological tissue changes and bacterial localization in chronic osteomyelitis with a focus on the pelvis. X-ray imaging was performed to follow the disease progression. Six weeks post infection, when osteomyelitis had manifested itself with a macroscopically visible bone deformation in the pelvis, we used two orthogonal methods, namely fluorescence imaging and label-free Raman spectroscopy, to characterise tissue changes on a microscopic scale and to localise bacteria in different tissue regions. Hematoxylin and eosin as well as Gram staining were performed as a reference method. We could detect all signs of a chronically florid tissue infection with osseous and soft tissue changes as well as with different inflammatory infiltrate patterns. Large lesions dominated in the investigated tissue samples. Bacteria were found to form abscesses and were distributed in high numbers in the lesion, where they could occasionally also be detected intracellularly. In addition, bacteria were found in lower numbers in surrounding muscle tissue and even in lower numbers in trabecular bone tissue. The Raman spectroscopic imaging revealed a metabolic state of the bacteria with reduced activity in agreement with small cell variants found in other studies. In conclusion, we present novel optical methods to characterise bone infections, including inflammatory host tissue reactions and bacterial adaptation. [ABSTRACT FROM AUTHOR]

L. monocytogenes is a widely used infection model for the research on pathogenesis and host defense against gram-positive intracellular bacteria. Emerging evidence indicates that posttranslational modifications play a critical role in the regulation of macroautophagy/autophagy. However, little is known about the posttranslational modifications of ATG7, the essential protein in the autophagy process. In this study, we demonstrated that the RING-type E3 ligase TRIM7/RNF90 positively regulated autophagosome accumulation by promoting the ubiquitination of ATG7 at K413, thereby affecting L. monocytogenes infection. TRIM7 expression was induced by a variety range of conditions, including starvation, rapamycin stimulation, and L. monocytogenes infection. TRIM7 deficiency in mice or cells resulted in elevated innate immune responses and increased L. monocytogenes infection. ATG7 was associated with TRIM7 and the positive regulatory role of TRIM7 in L. monocytogenes infection-, starvation- or rapamycin-induced autophagosome accumulation was suggested by TRIM7 deficiency, TRIM7 overexpression, and TRIM7 knockdown. Further mechanistic investigation indicated that TRIM7 promoted the K63-linked ubiquitination of ATG7 at K413 and ubiquitination at this site was required for the function of ATG7 in autophagy and L. monocytogenes infection. Thus, our findings suggested a new regulator in intracellular bacterial infection and autophagy, with a novel posttranslational modification targeting ATG7. This research may expand our understanding of host anti-bacterial defense and the role of autophagy in intracellular bacterial infection. Abbreviations: ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG10: autophagy related 10; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; Baf A1: bafilomycin A1; CQ: chloroquine; BMDC: bone marrow-derived dendritic cell; BMDM: bone marrow-derived macrophage; CFUs: colony-forming units; CXCL10/IP-10: C-X-C motif chemokine ligand 10; EBSS: Earle's balanced salt solution; ELISA: enzyme-linked immunosorbent assay; IFIT1/ISG56: interferon induced protein with tetratricopeptide repeats 1; IFNB/IFN-β: interferon beta; IL6: interleukin 6; IRF3, interferon regulatory factor 3; Lm: L. monocytogenes; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; PLA: proximity ligation assay; PMA: phorbol myristate acetate; PMA-THP1, PMA-differentiated THP1; PMs: peritoneal macrophages; PTMs: posttranslational modifications; STING1, stimulator of interferon response cGAMP interactor 1; TBK1, TANK binding kinase 1; TNF/TNF-α: tumor necrosis factor; TRIM7/RNF90: tripartite motif containing; Hainan Provincial Natural Science Foundation of China. [ABSTRACT FROM AUTHOR]

Intracellular bacteria serve as a problematic source of infection due to their ability to evade biological immune responses and the inability for conventional antibiotics to efficiently penetrate cellular membranes. Subsequently, new treatment approaches are urgently required to effectively eradicate intracellular pathogens residing within immune cells (e.g. macrophages). In this study, the poorly soluble and poorly permeable antibiotic, rifampicin, was re-purposed via micro-encapsulation within inulin-lipid hybrid (ILH) particles for the treatment of macrophages infected with small colony variants of Staphylococcus aureus (SCV S. aureus). Rifampicin-encapsulated ILH (Rif-ILH) microparticles were synthesized by spray drying a lipid nano-emulsion, with inulin dissolved throughout the aqueous phase and rifampicin pre-loaded within the lipid phase. Rif-ILH were strategically designed and engineered with pH-responsive properties to promote lysosomal drug release upon cellular internalization, while preventing premature rifampicin release in plasma-simulating media. The pH-responsiveness of Rif-ILH was controlled by the acid-mediated hydrolysis of the inulin coating, where exposure to acidic media simulating the lysosomal environment of macrophages triggered hydrolysis of the oligofructose chain and the subsequent diffusion of rifampicin from Rif-ILH. This pH-provoked release mechanism, as well as the ability for ILH microparticles to be more readily internalized by macrophages, was found to be influential in triggering a 2.9-fold increase in intracellular rifampicin concentration within infected macrophages, compared to the pure drug. The subsequent increase in exposure of intracellular pathogens to rifampicin leads to a ~ 2-log improvement in antibacterial activity for Rif-ILH, at a rifampicin dose of 2.5 µg/mL. Thus, the reduction in viability of intracellular SCV S. aureus, in the absence of cellular toxicity, is indicative of ILH microparticles serving as a unique approach for the safe and efficacious delivery of antibiotics to phagocytic cells for the treatment of intracellular infections. [ABSTRACT FROM AUTHOR]

Symbiosis with microbes is a ubiquitous phenomenon with a massive impact on all living organisms, shaping the world around us today. Theoretical and experimental studies show that vertical transmission of symbionts leads to the evolution of mutualistic traits, whereas horizontal transmission facilitates the emergence of parasitic features. However, these studies focused on phenotypic data, and we know little about underlying molecular changes at the genomic level. Here, we combined an experimental evolution approach with infection assays, genome resequencing, and global gene expression analysis to study the effect of transmission mode on an obligate intracellular bacterial symbiont. We show that a dramatic shift in the frequency of genetic variants, coupled with major changes in gene expression, allow the symbiont to alter its position in the parasitism-mutualism continuum depending on the mode of between-host transmission. We found that increased parasitism in horizontally transmitted chlamydiae residing in amoebae was a result of processes occurring at the infectious stage of the symbiont's developmental cycle. Specifically, genes involved in energy production required for extracellular survival and the type III secretion system-the symbiont's primary virulence mechanism-were significantly up-regulated. Our results identify the genomic and transcriptional dynamics sufficient to favor parasitic or mutualistic strategies.

Listeria monocytogenes can cause a life-threatening illness when the foodborne pathogen spreads beyond the intestinal tract to distant organs. Many aspects of the intestinal phase of L. monocytogenes pathogenesis remain unknown. Here, we present a foodborne infection model using C57BL/6 mice that have been pretreated with streptomycin. In this model, as few as 100 L. monocytogenes CFU were required to cause self-limiting enterocolitis, and systemic dissemination followed previously reported routes. Using this model, we report that listeriolysin O (LLO) and actin assembly-inducing protein (ActA), two critical virulence determinants, were necessary for intestinal pathology and systemic spread but were dispensable for intestinal growth. Sequence tag-based analysis of microbial populations (STAMP) was used to investigate the within-host population dynamics of wild-type and LLO-deficient strains. The wild-type bacterial population experienced severe bottlenecks over the course of infection, and by 5 days, the intestinal population was highly enriched for bacteria originating from the gallbladder. In contrast, LLO-deficient strains did not efficiently disseminate and gain access to the gallbladder, and the intestinal population remained diverse. These findings suggest that systemic spread and establishment of a bacterial reservoir in the gallbladder imparts an intraspecies advantage in intestinal occupancy. Since intestinal L. monocytogenes is ultimately released into the environment, within-host population bottlenecks may provide purifying selection of virulence genes.IMPORTANCEListeria monocytogenes maintains capabilities for free-living growth in the environment and for intracellular replication in a wide range of hosts, including livestock and humans. Here, we characterized an enterocolitis model of foodborne L. monocytogenes infection. This work highlights a multiorgan trafficking circuit and reveals a fitness advantage for bacteria that successfully complete this cycle. Because virulence factors play critical roles in systemic dissemination and multiple bottlenecks occur as the bacterial population colonizes different tissue sites, this multiorgan trafficking circuit likely provides purifying selection of virulence genes. This study also serves as a foundation for future work using the L. monocytogenes-induced enterocolitis model to investigate the biology of L. monocytogenes in the intestinal environment.

Priming of macrophages with interferon-gamma (IFNγ) or interleukin-4 (IL-4) leads to polarisation into pro-inflammatory or anti-inflammatory subtypes, which produce key enzymes such as inducible nitric oxide synthase (iNOS) and arginase 1 (ARG1), respectively, and in this way determine host responses to infection. Importantly, L-arginine is the substrate for both enzymes. ARG1 upregulation is associated with increased pathogen load in different infection models. However, while differentiation of macrophages with IL-4 impairs host resistance to the intracellular bacterium Salmonella enterica serovar Typhimurium (S.tm), little is known on the effects of IL-4 on unpolarised macrophages during infection. Therefore, bone-marrow-derived macrophages (BMDM) from C57BL/6N, Tie2Cre+/−ARG1fl/fl (KO), Tie2Cre−/−ARG1fl/fl (WT) mice were infected with S.tm in the undifferentiated state and then stimulated with IL-4 or IFNγ. In addition, BMDM of C57BL/6N mice were first polarised upon stimulation with IL-4 or IFNγ and then infected with S.tm. Interestingly, in contrast to polarisation of BMDM with IL-4 prior to infection, treatment of non-polarised S.tm-infected BMDM with IL-4 resulted in improved infection control whereas stimulation with IFNγ led to an increase in intracellular bacterial numbers compared to unstimulated controls. This effect of IL-4 was paralleled by decreased ARG1 levels and increased iNOS expression. Furthermore, the L-arginine pathway metabolites ornithine and polyamines were enriched in unpolarised cells infected with S.tm and stimulated with IL-4. Depletion of L-arginine reversed the protective effect of IL-4 toward infection control. Our data show that stimulation of S.tm-infected macrophages with IL-4 reduced bacterial multiplication via metabolic re-programming of L-arginine-dependent pathways. [ABSTRACT FROM AUTHOR]

Nocardia seriolae, an intracellular gram-positive pathogen, is prone to infecting immunocompromised and surface-damaged fish, causing serious losses to the aquaculture industry. Although a previous study has demonstrated that N. seriolae infects macrophages, the persistence of this bacterium in macrophages has not been well characterized. To address this gap, we used the macrophage cell line RAW264.7, to investigate the interactions between N. seriolae and macrophages and deciphered the intracellular survival mechanism of N. seriolae. Confocal and light microscopy revealed that N. seriolae entered macrophages 2 hours post-inoculation (hpi), were phagocytosed by macrophages at 4-8 hpi, and induced the formation of multinucleated macrophages by severe fusion at 12 hpi. Flow cytometry, evaluation of mitochondrial membrane potential, release of lactate dehydrogenase, and observation of the ultrastructure of macrophages revealed that apoptosis was induced in the early infection stage and inhibited in the middle and later periods of infection. Additionally, the expression of Bcl-2, Bax, Cyto-C, Caspase-3, Capase-8, and Caspase-9 was induced at 4 hpi, and then decreased at 6-8 hpi, illustrating that N. seriolae infection induces the activation of extrinsic and intrinsic apoptotic pathways in macrophages, followed by the inhibition of apoptosis to survive inside the cells. Furthermore, N. seriolae inhibits the production of reactive oxygen species and releases large amounts of nitric oxide, which persists in macrophages during infection. The present study provides the first comprehensive insight into the intracellular behavior of N. seriolae and its apoptotic effect on macrophages and may be important for understanding the pathogenicity of fish nocardiosis. [ABSTRACT FROM AUTHOR]

Identifying suitable animal models and standardizing preclinical methods are important for the generation, characterization, and development of new vaccines, including those against Francisella tularensis. Non-human primates represent an important animal model to evaluate tularemia vaccine efficacy, and the use of correlates of vaccine-induced protection may facilitate bridging immune responses from non-human primates to people. However, among small animals, Fischer 344 rats represent a valuable resource for initial studies to evaluate immune responses, to identify correlates of protection, and to screen novel vaccines. In this study, we performed a comparative analysis of three Fischer rat substrains to determine potential differences in immune responses, to evaluate methods used to quantify potential correlates of protection, and to evaluate protection after vaccination. To this end, we took advantage of data previously generated using one of the rat substrains by evaluating two live vaccines, LVS and F. tularensis SchuS4-ΔclpB (ΔclpB). We compared immune responses after primary vaccination, adaptive immune responses upon re-stimulation of leukocytes in vitro, and sensitivity to aerosol challenge. Despite some detectable differences, the results highlight the similarity of immune responses to tularemia vaccines and challenge outcomes between the three substrains, indicating that all offer acceptable and comparable approaches as animal models to study Francisella infection and immunity.

IntroductionTularemia is mainly caused by Francisella tularensis (Ft) subsp. tularensis (Ftt) and Ft subsp. holarctica (Ftt) in humans and in more than 200 animal species including rabbits and hares. Human clinical manifestations depend on the route of infection and range from flu-like symptoms to severe pneumonia with a mortality rate up to 60% without treatment. So far, only 2D cell culture and animal models are used to study Francisella virulence, but the gained results are transferable to human infections only to a certain extent.MethodIn this study, we firstly established an ex vivo human lung tissue infection model using different Francisella strains: Ftt Life Vaccine Strain (LVS), Ftt LVS ΔiglC, Ftt human clinical isolate A-660 and a German environmental Francisella species strain W12-1067 (F-W12). Human lung tissue was used to determine the colony forming units and to detect infected cell types by using spectral immunofluorescence and electron microscopy. Chemokine and cytokine levels were measured in culture supernatants.ResultsOnly LVS and A-660 were able to grow within the human lung explants, whereas LVS ΔiglC and F-W12 did not replicate. Using human lung tissue, we observed a greater increase of bacterial load per explant for patient isolate A-660 compared to LVS, whereas a similar replication of both strains was observed in cell culture models with human macrophages. Alveolar macrophages were mainly infected in human lung tissue, but Ftt was also sporadically detected within white blood cells. Although Ftt replicated within lung tissue, an overall low induction of pro-inflammatory cytokines and chemokines was observed. A-660-infected lung explants secreted slightly less of IL-1β, MCP-1, IP-10 and IL-6 compared to Ftt LVS-infected explants, suggesting a more repressed immune response for patient isolate A-660. When LVS and A-660 were used for simultaneous co-infections, only the ex vivo model reflected the less virulent phenotype of LVS, as it was outcompeted by A-660.ConclusionWe successfully implemented an ex vivo infection model using human lung tissue for Francisella. The model delivers considerable advantages and is able to discriminate virulent Francisella from less- or non-virulent strains and can be used to investigate the role of specific virulence factors.

The amoeba-resistant bacterium Legionella pneumophila causes Legionnaires’ disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated Legionella-containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoeba Dictyostelium discoideum. Dually fluorescence-labeled D. discoideum producing LCV and LD markers revealed that Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δsey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila fatty acid transporter FadL were implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.

FISH techniques have been applied for the visualization and identification of intracellular bacteria in companion animal species. Most frequently, these techniques have focused on the identification of adhesive-invasive Escherichia coli in gastrointestinal disease, although various other organisms have been identified in inflammatory or neoplastic gastrointestinal disease. Previous studies have investigated a potential role of Helicobacter spp. in inflammatory gastrointestinal and hepatic conditions. Other studies evaluating the role of infectious organisms in hepatopathies have received some attention with mixed results. FISH techniques using both eubacterial and species-specific probes have been applied in inflammatory cardiovascular, urinary, and cutaneous diseases to screen for intracellular bacteria. This review summarizes the results of these studies.

During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factors that regulate killing by L. pneumophila. The screens reveal known host factors hijacked by L. pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L. pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators of phagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L. pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L. pneumophila pathogenesis and mammalian cell function.

Pseudomonas aeruginosa is among bacterial pathogens capable of twitching motility, a form of surface-associated movement dependent on type IV pili (T4P). Previously, we showed that T4P and twitching were required for P. aeruginosa to cause disease in a murine model of corneal infection, to traverse human corneal epithelial multilayers, and to efficiently exit invaded epithelial cells. Here, we used live wide-field fluorescent imaging combined with quantitative image analysis to explore how twitching contributes to epithelial cell egress. Results using time-lapse imaging of cells infected with wild-type PAO1 showed that cytoplasmic bacteria slowly disseminated throughout the cytosol at a median speed of >0.05 μm s-1 while dividing intracellularly. Similar results were obtained with flagellin (fliC) and flagellum assembly (flhA) mutants, thereby excluding swimming, swarming, and sliding as mechanisms. In contrast, pilA mutants (lacking T4P) and pilT mutants (twitching motility defective) appeared stationary and accumulated in expanding aggregates during intracellular division. Transmission electron microscopy confirmed that these mutants were not trapped within membrane-bound cytosolic compartments. For the wild type, dissemination in the cytosol was not prevented by the depolymerization of actin filaments using latrunculin A and/or the disruption of microtubules using nocodazole. Together, these findings illustrate a novel form of intracellular bacterial motility differing from previously described mechanisms in being directly driven by bacterial motility appendages (T4P) and not depending on polymerized host actin or microtubules.IMPORTANCE Host cell invasion can contribute to disease pathogenesis by the opportunistic pathogen Pseudomonas aeruginosa Previously, we showed that the type III secretion system (T3SS) of invasive P. aeruginosa strains modulates cell entry and subsequent escape from vacuolar trafficking to host lysosomes. However, we also showed that mutants lacking either type IV pili (T4P) or T4P-dependent twitching motility (i) were defective in traversing cell multilayers, (ii) caused less pathology in vivo, and (iii) had a reduced capacity to exit invaded cells. Here, we report that after vacuolar escape, intracellular P. aeruginosa can use T4P-dependent twitching motility to disseminate throughout the host cell cytoplasm. We further show that this strategy for intracellular dissemination does not depend on flagellin and resists both host actin and host microtubule disruption. This differs from mechanisms used by previously studied pathogens that utilize either host actin or microtubules for intracellular dissemination independently of microbe motility appendages.