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Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M -1 s -1 . The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M -1 s -1 . Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 10 4  M -1 s -1 to 20.3 ± 4.3 × 10 4  M -1 s -1 . We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 10 4  M -1 s -1 ) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 10 5  M -1 s -1 ). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Therapeutic manipulation of the gut microbiota holds great potential for human health. The mechanisms bacteria use to colonize the gut therefore present valuable targets for clinical intervention. We now report that bacteria use phase separation to enhance fitness in the mammalian gut. We establish that the intrinsically disordered region (IDR) of the broadly and highly conserved transcription termination factor Rho is necessary and sufficient for phase separation in vivo and in vitro in the human commensal Bacteroides thetaiotaomicron . Phase separation increases transcription termination by Rho in an IDR-dependent manner. Moreover, the IDR is critical for gene regulation in the gut. Our findings expose phase separation as vital for host-commensal bacteria interactions and relevant for novel clinical applications.

The multifunctional GSDMB protein is an important molecule in human immunity. The pyroptotic and bactericidal activity of GSDMB is a host response to infection by the bacterial pathogen Shigella flexneri, which employs the virulence effector IpaH7.8 to ubiquitinate and target GSDMB for proteasome-dependent degradation. Furthermore, IpaH7.8 selectively targets human but not mouse GSDMD, suggesting a non-canonical mechanism of substrate selection. Here, we report the crystal structure of GSDMB in complex with IpaH7.8. Together with biochemical and functional studies, we identify the potential membrane engagement sites of GSDMB, revealing general and unique features of gasdermin proteins in membrane recognition. We further illuminate how IpaH7.8 interacts with GSDMB, and delineate the mechanism by which IpaH7.8 ubiquitinates and suppresses GSDMB. Notably, guided by our structural model, we demonstrate that two residues in the α1-α2 loop make the mouse GSDMD invulnerable to IpaH7.8-mediated degradation. These findings provide insights into the versatile functions of GSDMB, which could open new avenues for therapeutic interventions for diseases, including cancers and bacterial infections., (© 2023. The Author(s).)

Legionella pneumophila is a Gram-negative bacterium found in natural and man-made water systems where it replicates within amoebas and ciliates. In humans, once inside the lungs, L. pneumophila replicates in alveolar macrophages and causes Legionnaires' disease, a severe pneumonia. The Icm/Dot type IVb secretion system is a major virulence factor required for intracellular multiplication. The Icm/Dot system allows the secretion of effectors into the cytoplasm of the host cell. These effectors modify host cell vesicular trafficking and prevent maturation of the phagosome. The innate immune response is crucial in restricting L. pneumophila proliferation. TNF-α is one of the major cytokines involved in this process as it renders macrophages more resistant to L. pneumophila infection and induces apoptosis of L. pneumophila -infected macrophages. Tail-specific proteases (Tsp) are involved in tolerating thermal stress and in virulence. We have previously characterized the Tsp encoded by L. pneumophila , showing that it is important for surviving thermal stress and for infection of amoeba when a temperature change occurs during infection. Here, we demonstrated that Tsp is required for intracellular multiplication in macrophages. Absence of tsp is associated with higher production of TNF-α by macrophages in response to L. pneumophila infection. This effect is independent of the Icm/Dot secretion system.

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator ( agr ) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of agr QS (MroQ), an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

Brucella canis is responsible for canine brucellosis, a neglected zoonotic disease. The omp25 gene has been described as an important marker for Brucella intra-species differentiation, in addition to the ability to interact with the host immune system. Therefore, this study investigated the omp25 sequence from B. canis strains associated to a phylogenetic characterization and the unveiling of the molecular structure. In vitro analyses comprised DNA extraction, PCR, and sequencing of omp25 from 19 B. canis strains. Moreover, in silico analyses were performed at nucleotide level for phylogenetic characterization and evolutionary history of B. canis omp25 gene; and in amino acid level including modeling, dynamics, and epitope prediction of B. canis Omp25 protein. Here, we identified a new mutation, L109P, which diverges the worldwide omp25 sequences in two large branches. Interestingly, this mutation appears to have epidemiology importance, based on a geographical distribution of B. canis strains. Structural and molecular dynamics analyses of Omp25 revealed that Omp25 L109P does not sustain its native β-barrel. Likewise, the conformation of B-cell epitope on the mutated region was changed in Omp25 L109P protein. Even without an evolutive marker, the new identified mutation appears to affect the basic function of B. canis Omp25 protein, which could indicate virulence adaptation for some B. canis strains in a context of geographical disposition., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Serratia marcescens is an opportunistic bacterium that infects a wide range of hosts including humans. It is a potent pathogen in a septic injury model of Drosophila melanogaster since a few bacteria directly injected in the body cavity kill the insect within a day. In contrast, flies do not succumb to ingested bacteria for days even though some bacteria cross the intestinal barrier into the hemolymph within hours. The mechanisms by which S. marcescens attacks enterocytes and damages the intestinal epithelium remain uncharacterized. To better understand intestinal infections, we performed a genetic screen for loss of virulence of ingested S. marcescens and identified FliR, a structural component of the flagellum, as a virulence factor. Next, we compared the virulence of two flagellum mutants fliR and flhD in two distinct S. marcescens strains. Both genes are required for S. marcescens to escape the gut lumen into the hemocoel, indicating that the flagellum plays an important role for the passage of bacteria through the intestinal barrier. Unexpectedly, fliR but not flhD is involved in S. marcescens-mediated damages of the intestinal epithelium that ultimately contribute to the demise of the host. Our results therefore suggest a flagellum-independent role for fliR in bacterial virulence., (© 2022. The Author(s).)

Mycobacterium tuberculosis has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes desA1 and desA2 , controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR -null mutant of M. smegmatis exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon madR deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of β-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of desA1 / desA2 is relieved upon binding of saturated acyl-CoAs of chain length C 16 to C 24 , while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD have proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular, and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was relatively enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host-pathogen interaction. Compared to the other subcellular fractions, the MVs were also relatively enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs., (© 2022. The Author(s).)

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram-negative bacterium with a large genome of ∼8 Mb that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. We previously identified an immunoreactive acidic protein encoded on replicon 3, BCAS0292. Deletion of the BCAS0292 gene significantly altered the abundance of 979 proteins by 1.5-fold or more; 19 proteins became undetectable while 545 proteins showed ≥1.5-fold reduced abundance, suggesting the BCAS0292 protein is a global regulator. Moreover, the ∆BCAS0292 mutant showed a range of pleiotropic effects: virulence and host-cell attachment were reduced, antibiotic susceptibility was altered, and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. In silico prediction of its three-dimensional structure revealed BCAS0292 presents a dimeric β-structure with a negative surface charge. The ΔBCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. Three proteins were identified in pull-downs with FLAG-tagged BCAS0292, including the Histone H1-like protein, HctB, which is recognized as a global transcriptional regulator. We propose that BCAS0292 protein, which we have named Burkholderia negatively surface-charged regulatory protein 1 (Bnr1), acts as a DNA-mimic and binds to DNA-binding proteins, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments., (© 2022 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Staphylococcus aureus is an opportunistic, pathogenic bacteria that causes significant morbidity and mortality. As antibiotic resistance by S. aureus continues to be a serious concern, developing novel drug therapies to combat these infections is vital. Quorum sensing inhibitors (QSI) dampen S. aureus virulence and facilitate clearance by the host immune system by blocking quorum sensing signaling that promotes upregulation of virulence genes controlled by the accessory gene regulator (agr) operon. While QSIs have shown therapeutic promise in mouse models of S. aureus skin infection, their further development has been hampered by the suggestion that agr inhibition promotes biofilm formation. In these studies, we investigated the relationship between agr function and biofilm growth across various S. aureus strains and experimental conditions, including in a mouse model of implant-associated infection. We found that agr deletion was associated with the presence of increased biofilm only under narrow in vitro conditions and, crucially, was not associated with enhanced biofilm development or enhanced morbidity in vivo., (© 2022. The Author(s).)

Roles for the non-coding small RNA RyhB in quorum-sensing and iron-dependent gene modulation in the human pathogen V. vulnificus were assessed in this study. Both the quorum sensing master regulator SmcR and the Fur-iron complex were observed to bind to the region upstream of the non-coding small RNA RyhB gene to repress expression, which suggests that RyhB is associated with both quorum-sensing and iron-dependent signaling in this pathogen. We found that expression of LuxS, which is responsible for the biosynthesis of autoinducer-2 (AI-2), was higher in wild type than in a ryhB-deletion isotype. RyhB binds directly to the 5'-UTR (untranslated region) of the luxS transcript to form a heteroduplex, which not only stabilizes luxS mRNA but also disrupts the secondary structure that normally obscures the translational start codon and thereby allows translation of LuxS to begin. The binding of RyhB to luxS mRNA requires the chaperone protein Hfq, which stabilizes RyhB. These results demonstrate that the small RNA RyhB is a key element associated with feedback control of AI-2 production, and that it inhibits quorum-sensing signaling in an iron-dependent manner. This study, taken together with previous studies, shows that iron availability and cell density signals are funneled to SmcR and RyhB, and that these regulators coordinate cognate signal pathways that result in the proper balance of protein expression in response to environmental conditions., (© 2022. The Author(s).)

The insecticidal Vip3 proteins, secreted by Bacillus thuringiensis ( Bt ) during its vegetative growth phase, are currently used in Bt crops to control insect pests, and are genetically distinct from known insecticidal Cry proteins. Compared with Cry toxins, the mechanisms of Vip3 toxins are still poorly understood. Here, the responses of Spodoptera frugiperda larvae after Vip3Aa challenge are characterized. Using an integrative analysis of transcriptomics and proteomics, we found that Vip3Aa has enormous implications for various pathways. The downregulated genes and proteins were mainly enriched in metabolic pathways, including the insect hormone synthesis pathway, whereas the upregulated genes and proteins were mainly involved in the caspase-mediated apoptosis pathway, along with the MAPK signaling and endocytosis pathways. Moreover, we also identified some important candidate genes involved in apoptosis and MAPKs. The present study shows that exposure of S. frugiperda larvae to Vip3Aa activates apoptosis pathways, leading to cell death. The results will promote our understanding of the host response process to the Vip3Aa, and help us to better understand the mode of action of Vip3A toxins.

Carbon Storage Regulator A (CsrA) is a well-characterized post-transcriptional global regulator that plays a critical role in response to environmental changes in many bacteria. CsrA has been reported to regulate several metabolic pathways, motility, biofilm formation, and virulence-associated genes. The role of csrA in Leptospira spp., which are able to survive in different environmental niches and infect a wide variety of reservoir hosts, has not been characterized. To investigate the role of csrA as a gene regulator in Leptospira, we generated a L. biflexa csrA deletion mutant (ΔcsrA) and csrA overexpressing Leptospira strains. The ΔcsrA L. biflexa displayed poor growth under starvation conditions. RNA sequencing revealed that in rich medium only a few genes, including the gene encoding the flagellar filament protein FlaB3, were differentially expressed in the ΔcsrA mutant. In contrast, 575 transcripts were differentially expressed when csrA was overexpressed in L. biflexa. Electrophoretic mobility shift assay (EMSA) confirmed the RNA-seq data in the ΔcsrA mutant, showing direct binding of recombinant CsrA to flaB3 mRNA. In the pathogen L. interrogans, we were not able to generate a csrA mutant. We therefore decided to overexpress csrA in L. interrogans. In contrast to the overexpressing strain of L. biflexa, the overexpressing L. interrogans strain had poor motility on soft agar. The overexpressing strain of L. interrogans also showed significant upregulation of the flagellin flaB1, flaB2, and flaB4. The interaction of L. interrogans rCsrA and flaB4 was confirmed by EMSA. Our results demonstrated that CsrA may function as a global regulator in Leptospira spp. under certain conditions that cause csrA overexpression. Interestingly, the mechanisms of action and gene targets of CsrA may be different between non-pathogenic and pathogenic Leptospira strains., Competing Interests: The authors have declared that no competing interests exist.

Cronobacter sakazakii is a food-borne, conditionally pathogenic bacterium that mainly infects neonates, especially premature infants. Previous studies have indicated that an important route of infection for C. sakazakii is through infant formula, suggesting a high stress resistance of the bacterium. RpoS is a σ-factor that is closely related to the bacterial resistance mechanisms. In this study, a C. sakazakii BAA894 model strain was used. An rpoS -deficient mutant strain Δ rpos was constructed using Red homologous recombination, and the differences between the mutant and the wild-type strains were compared. To investigate the functions of the rpoS gene, the membrane formation and cell wall properties of the strains were studied, and the tolerance of each strain to acid, osmotic pressure, desiccation, and drug resistance were compared. The results showed that the membrane formation ability in the mutant strain was increased, auto-aggregation was enhanced, motility, acid resistance and hyperosmotic resistance were alternated to different degrees, and desiccation resistance was stronger than observed in the wild type grown in LB medium but weaker than the wild type cultured in M9 medium. These results showed that rpoS is involved in environmental stress resistance in C. sakazakii BAA894. Finally, transcriptome analysis verified that the deletion of the rpoS gene caused differential expression of resistance-related genes and instigated changes in related metabolic pathways. These messenger RNA results were consistent with the functional experimental results and help explain the phenotypic changes observed in the mutant strain.

We studied the effect of bacterial wall peptidoglycan of 7 bacterial species on the competitive properties of human-associated microorganisms. Addition of peptidoglycan to the culture medium did not change the growth characteristics of the test cultures; however, an increase in the antagonism and hydrophobicity of Bifidobacterium sp. and Enterococcus sp. was observed, while the effect on enterobacteria was predominantly indifferent or inhibitory. The effect did not depend much on the source of peptidoglycan and was equally manifested on both indigenous and probiotic strains. The observed new property of peptidoglycan indicates its participation in the formation and functioning of microbiota. The obtained data on the regulation of the properties of microorganisms provide new possibilities for the correction and maintenance of host homeostasis through host-associated microbiota., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Resistance exercise (RE) with blood flow restriction (BFR) is recognized as a beneficial strategy in increasing skeletal muscle mass and strength. However, the effects of BFR on changes in perceptual parameters, particularly those related to exercise adherence, induced by RE are not completely understood. In this study, we examined the exercise adherence-related perceptual responses to low-load BFR-RE. Sixteen young males performed both BFR and non-BFR (NBFR) sessions in a crossover design. The bilateral knee extensor low-load RE was performed with a standard BFR-RE protocol, consisting of four sets (total 75 repetitions), using 20% of one-repetition maximum. BFR-RE was performed with 200 mmHg pressure cuffs placed around the proximal region of the thighs. NBFR-RE was performed without pressure cuffs. The ratings of perceived exertion and leg discomfort measured using the Borg's Scales were higher for BFR-RE session than for NBFR-RE session (both p < 0.001 for interaction effect). The Feeling Scale-measured affect and Task Motivation Scale-measured task motivation were lower for BFR-RE session than for NBFR-RE session (both p < 0.05 for interaction effect); by contrast, the Numerical Rating Scale-measured perceived pain was higher for BFR-RE session than for NBFR-RE session (p < 0.001 for interaction effect). The Physical Activity Enjoyment Scale-measured enjoyment immediately after RE was lower with BFR than with NBFR (p < 0.001). These findings suggest that BFR exacerbates the exercise adherence-related perceptual responses to low-load RE in young males. Therefore, further studies are needed to develop effective strategies that minimize the BFR-RE-induced negative effects on perceptual responses., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Prodigiosin is a tripyrrole red secondary metabolite synthesized by many microorganisms, including Serratia marcescens. In this study, we found that the deletion of the gene of sensor kinase CpxA dramatically decreased the prodigiosin production, while the deletion of the gene of the response regulator CpxR or both genes of CpxRA has no effect on prodigiosin production, the kinase function of CpxA is not essential for its regulation on prodigiosin production while the phosphorylation site of CpxR is required. We further demonstrated that the CpxA regulates the prodigiosin biosynthesis at the transcriptional level and the phosphatase activity of CpxA plays vital roles in the regulation of prodigiosin biosynthesis. Finally, we proposed that CpxR/A regulates the prodigiosin biosynthesis by negative control and the phosphorylation level of CpxR may determine the positive or negative control of the genes it regulated., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Multi-drug resistance (MDR) bacterial pathogens pose a threat to global health and warrant the discovery of new therapeutic molecules, particularly those that can neutralize their virulence and stop the evolution of new resistant mechanisms. The superbug nosocomial pathogen, Pseudomonas aeruginosa , uses a multiple virulence factor regulator (MvfR) to regulate the expression of multiple virulence proteins during acute and persistent infections. The present study targeted MvfR with the intention of designing novel anti-virulent compounds, which will function in two ways: first, they will block the virulence and pathogenesis P. aeruginosa by disrupting the quorum-sensing network of the bacteria, and second, they will stop the evolution of new resistant mechanisms. A structure-based virtual screening (SBVS) method was used to screen druglike compounds from the Asinex antibacterial library (~5968 molecules) and the comprehensive marine natural products database (CMNPD) (~32 thousand compounds), against the ligand-binding domain (LBD) of MvfR, to identify molecules that show high binding potential for the relevant pocket. In this way, two compounds were identified: Top-1 (4-((carbamoyloxy)methyl)-10,10-dihydroxy-2,6-diiminiodecahydropyrrolo[1,2-c]purin-9-yl sulfate) and Top-2 (10,10-dihydroxy-2,6-diiminio-4-(((sulfonatocarbamoyl)oxy)methyl)decahydropyrrolo[1,2-c]purin-9-yl sulfate), in contrast to the co-crystallized M64 control. Both of the screened leads were found to show deep pocket binding and interactions with several key residues through a network of hydrophobic and hydrophilic interactions. The docking results were validated by a long run of 200 ns of molecular dynamics simulation and MM-PB/GBSA binding free energies. All of these analyses confirmed the presence of strong complex formation and rigorous intermolecular interactions. An additional analysis of normal mode entropy and a WaterSwap assay were also performed to complement the aforementioned studies. Lastly, the compounds were found to show an acceptable range of pharmacokinetic properties, making both compounds potential candidates for further experimental studies to decipher their real biological potency.

Barnase is an extracellular ribonuclease secreted by Bacillus amyloliquefaciens that was originally studied as a small stable enzyme with robust folding. The identification of barnase intracellular inhibitor barstar led to the discovery of an incredibly strong protein-protein interaction. Together, barnase and barstar provide a fully genetically encoded toxin-antitoxin pair having an extremely low dissociation constant. Moreover, compared to other dimerization systems, the barnase-barstar module provides the exact one-to-one ratio of the complex components and possesses high stability of each component in a complex and high solubility in aqueous solutions without self-aggregation. The unique properties of barnase and barstar allow the application of this pair for the engineering of different variants of targeted anticancer compounds and cytotoxic supramolecular complexes. Using barnase in suicide gene therapy has also found its niche in anticancer therapy. The application of barnase and barstar in contemporary experimental cancer therapy is reflected in the review.

Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections., Competing Interests: The authors declare no competing interest.

Salmonella Infantis has emerged as a major clinical pathogen causing gastroenteritis worldwide in recent years. As an intracellular pathogen, Salmonella has evolved to manipulate and benefit from the cell death signaling pathway. In this study, we discovered that S . Infantis inhibited apoptosis of infected Caco-2 cells by phosphorylating Akt. Notably, Akt phosphorylation was observed in a discontinuous manner: immediately 0.5 h after the invasion, then before peak cytosolic replication. Single-cell analysis revealed that the second phase was only induced by cytosolic hyper-replicating bacteria at 3-4 hpi. Next, Akt-mediated apoptosis inhibition was found to be initiated by Salmonella SopB. Furthermore, Akt phosphorylation increased mitochondrial localization of Bcl-2 to prevent Bax oligomerization on the mitochondrial membrane, maintaining the mitochondrial network homeostasis to resist apoptosis. In addition, S . Infantis induced pyroptosis, as evidenced by increased caspase-1 (p10) and GSDMS-N levels. In contrast, cells infected with the Δ SopB strain displayed faster but less severe pyroptosis and had less bacterial load. The results indicated that S . Infantis SopB -mediated Akt phosphorylation delayed pyroptosis, but aggravated its severity. The wild-type strain also caused more severe diarrhea and intestinal inflammatory damage than the Δ SopB strain in mice. These findings revealed that S . Infantis delayed the cells' death by intermittent activation of Akt, allowing sufficient time for replication, thereby causing more severe inflammation., 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 © 2021 Chu, Li, Liu, Yuan, Chen, Zhu and Wang.)

The summarized amalgam of internal relaxation modulations and external forces like pH, temperature, and solvent conditions determine the protein structure, stability, and function. In a free-energy landscape, although conformers are arranged in vertical hierarchy, there exist several adjacent parallel sets with conformers occupying equivalent energy cleft. Such conformational states are pre-requisites for the functioning of proteins that have oscillating environmental conditions. As these conformational changes have utterly small re-arrangements, nuclear magnetic resonance (NMR) spectroscopy is unique in elucidating the structure-dynamics-stability-function relationships for such conformations. Helicobacter pylori survives and causes gastric cancer at extremely low pH also. However, least is known as to how the genome of the pathogen is protected from reactive oxygen species (ROS) scavenging in the gut at low pH under acidic stress. In the current study, biophysical characteristics of H. pylori DNA binding protein (Hup) have been elucidated at pH 2 using a combination of circular dichroism, fluorescence, NMR spectroscopy, and molecular dynamics simulations. Interestingly, the protein was found to have conserved structural features, differential backbone dynamics, enhanced stability, and DNA binding ability at low pH as well. In summary, the study suggests the partaking of Hup protein even at low pH in DNA protection for maintaining the genome integrity.

Many bacteria can migrate from a free-living, planktonic state to an attached, biofilm existence. One factor regulating this transition in the facultative plant pathogen Agrobacterium tumefaciens is the ExoR-ChvG-ChvI system. Periplasmic ExoR regulates the activity of the ChvG-ChvI two-component system in response to environmental stress, most notably low pH. ChvI impacts hundreds of genes, including those required for type VI secretion, virulence, biofilm formation, and flagellar motility. Previous studies revealed that activated ChvG-ChvI represses expression of most of class II and class III flagellar biogenesis genes, but not the master motility regulator genes visN, visR, and rem. In this study, we characterized the integration of the ExoR-ChvG-ChvI and VisNR-Rem pathways. We isolated motile suppressors of the non-motile ΔexoR mutant and thereby identified the previously unannotated mirA gene encoding a 76 amino acid protein. We report that the MirA protein interacts directly with the Rem DNA-binding domain, sequestering Rem and preventing motility gene activation. The ChvG-ChvI pathway activates mirA expression and elevated mirA is sufficient to block motility. This study reveals how the ExoR-ChvG-ChvI pathway prevents flagellar motility in A. tumefaciens. MirA is also conserved among other members of the Rhizobiales suggesting similar mechanisms of motility regulation., (© 2021 John Wiley & Sons Ltd.)

Bacteria form biofilms for their protection against environmental stress and produce virulence factors within the biofilm. Biofilm formation in acidified environments is regulated by a two-component system, as shown by studies on isogenic mutants of the sensor protein of the two-component regulatory system in Streptococcus pyogenes. In this study, we found that the LiaS histidine kinase sensor mediates biofilm production and pilus expression in an acidified environment through glucose fermentation. The liaS isogenic mutant produced biofilms in a culture acidified by hydrochloric acid but not glucose, suggesting that the acidified environment is sensed by another protein. In addition, the trxS isogenic mutant could not produce biofilms or activate the mga promoter in an acidified environment. Mass spectrometry analysis showed that TrxS regulates M protein, consistent with the transcriptional regulation of emm , which encodes M protein. Our results demonstrate that biofilm production during environmental acidification is directly under the control of TrxS.

The mechanisms by which bacteria sense the host environment and alter gene expression are poorly understood. LiaFSR is a gene regulatory system unique to Gram-positive bacteria, including group A Streptococcus (GAS), and responds to cell envelope stress. We previously showed that LiaF acts as an inhibitor to LiaFSR activation in GAS. To better understand gene regulation associated with LiaFSR activation, we performed RNA sequencing on isogenic deletion mutants fixed in a LiaFSR "always on" (Δ liaF ) or "always off" (Δ liaR ) state. Transcriptome analyses of Δ liaF and Δ liaR in GAS showed near perfect inverse correlation, including the gene encoding the global transcriptional regulator SpxA2. In addition, mutant transcriptomes included genes encoding multiple virulence factors and showed substantial overlap with the CovRS regulon. Chromatin immunoprecipitation quantitative PCR demonstrated direct spxA2 gene regulation following activation of the response regulator, LiaR. High SpxA2 levels as a result of LiaFSR activation were directly correlated with increased CovR-regulated virulence gene transcription. Furthermore, consistent with known virulence gene repression by phosphorylated CovR, elevated SpxA2 levels were inversely correlated with CovR phosphorylation. Despite increased transcription of several virulence factors, Δ liaF (high SpxA2) exhibited a paradoxical virulence phenotype in both in vivo mouse and ex vivo human blood models of disease. Likewise, despite decreased virulence factor transcription with Δ liaR (low SpxA2), increased virulence was observed in an in vivo mouse model of disease-a phenotype attributable, in part, to known SpxA2-associated speB transcription. Our findings provide evidence of a critical role of LiaFSR in sensing the host environment and suggest a potential mechanism for gene regulatory system cross talk shared by many Gram-positive pathogens.

The ability of Enterococcus faecalis to colonize host anatomical sites is dependent on its adaptive response to host conditions. Three glycosyl hydrolase gene clusters, each belonging to glycosyl hydrolase family 18 (GH18) ( ef0114 , ef0361 , and ef2863 ), in E. faecalis were previously found to be upregulated under glucose-limiting conditions. The GH18 catalytic domain is present in proteins that are classified as either chitinases or β-1,4 endo-β- N -acetylglucosaminidases (ENGases) based on their β-1,4 endo- N -acetyl-β-d-glucosaminidase activity, and ENGase activity is commonly associated with cleaving N-linked glycoprotein, an abundant glycan structure on host epithelial surfaces. Here, we show that all three hydrolases are negatively regulated by the transcriptional regulator carbon catabolite protein A (CcpA). Additionally, we demonstrate that a constitutively active CcpA variant represses the expression of CcpA-regulated genes irrespective of glucose availability. Previous studies showed that the GH18 catalytic domains of EndoE (EF0114) and EfEndo18A (EF2863) were capable of deglycosylating RNase B, a model high-mannose-type glycoprotein. However, it remained uncertain which glycosidase is primarily responsible for the deglycosylation of high-mannose-type glycoproteins. In this study, we show by mutation analysis as well as a dose-dependent analysis of recombinant protein expression that EfEndo18A is primarily responsible for deglycosylating high-mannose glycoproteins and that the glycans removed by EfEndo18A support growth under nutrient-limiting conditions in vitro . In contrast, IgG is representative of a complex-type glycoprotein, and we demonstrate that the GH18 domain of EndoE is primarily responsible for the removal of this glycan decoration. Finally, our data highlight the combined contribution of glycosidases to the virulence of E. faecalis in vivo.

Group A Streptoccocus (GAS) is among the most diverse of all human pathogens, responsible for a range of clinical manifestations, from mild superficial infections such as pharyngitis to serious invasive infections such as necrotising fasciitis and sepsis. The drivers of these different disease phenotypes are not known. The GAS cholesterol-dependent cytolysin, Streptolysin O (SLO), has well established cell and tissue destructive activity. We investigated the role of SLO in determining disease outcome in vivo, by using two different clinical lineages; the recently emerged hypervirulent outbreak emm type 32.2 strains, which result in sepsis, and the emm type 1.0 strains which cause septic arthritis. Using clinically relevant in vivo mouse models of sepsis and a novel septic arthritis model, we found that the amount and activity of SLO was vital in determining the course of infection. The emm type 32.2 strain produced large quantities of highly haemolytic SLO that resulted in rapid development of sepsis. By contrast, the reduced concentration and lower haemolytic activity of emm type 1.0 SLO led to translocation of bacteria from blood to joints. Importantly, sepsis associated strains that were attenuated by deletion or inhibition of SLO, then also translocated to the joint, confirming the key role of SLO in determining infection niche. Our findings demonstrate that SLO is key to in vivo phenotype and disease outcome. Careful consideration should be given to novel therapy or vaccination strategies that target SLO. Whilst neutralising SLO activity may reduce severe invasive disease, it has the potential to promote chronic inflammatory conditions such as septic arthritis., (© 2021. The Author(s).)

Virulence of the neonatal pathogen Group B Streptococcus is under the control of the master regulator CovR. Inactivation of CovR is associated with large-scale transcriptome remodeling and impairs almost every step of the interaction between the pathogen and the host. However, transcriptome analyses suggested a plasticity of the CovR signaling pathway in clinical isolates leading to phenotypic heterogeneity in the bacterial population. In this study, we characterized the CovR regulatory network in a strain representative of the CC-17 hypervirulent lineage responsible of the majority of neonatal meningitis. Transcriptome and genome-wide binding analysis reveal the architecture of the CovR network characterized by the direct repression of a large array of virulence-associated genes and the extent of co-regulation at specific loci. Comparative functional analysis of the signaling network links strain-specificities to the regulation of the pan-genome, including the two specific hypervirulent adhesins and horizontally acquired genes, to mutations in CovR-regulated promoters, and to variability in CovR activation by phosphorylation. This regulatory adaptation occurs at the level of genes, promoters, and of CovR itself, and allows to globally reshape the expression of virulence genes. Overall, our results reveal the direct, coordinated, and strain-specific regulation of virulence genes by the master regulator CovR and suggest that the intra-species evolution of the signaling network is as important as the expression of specific virulence factors in the emergence of clone associated with specific diseases., Competing Interests: The authors have declared that no competing interests exist.

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cyanobacteria synthesize type IV pili, which are known to be essential for motility, adhesion and natural competence. They consist of long flexible fibers that are primarily composed of the major pilin PilA1 in Synechocystis sp. PCC 6803. In addition, Synechocystis encodes less abundant pilin-like proteins, which are known as minor pilins. In this study, we show that the minor pilin PilA5 is essential for natural transformation but is dispensable for motility and flocculation. In contrast, a set of minor pilins encoded by the pilA9-slr2019 transcriptional unit are necessary for motility but are dispensable for natural transformation. Neither pilA5-pilA6 nor pilA9-slr2019 are essential for pilus assembly as mutant strains showed type IV pili on the cell surface. Three further gene products with similarity to PilX-like minor pilins have a function in flocculation of Synechocystis. The results of our study indicate that different minor pilins facilitate distinct pilus functions. Further, our microarray analysis demonstrated that the transcription levels of the minor pilin genes change in response to surface contact. A total of 122 genes were determined to have altered transcription between planktonic and surface growth, including several plasmid genes which are involved exopolysaccharide synthesis and the formation of bloom-like aggregates., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Efficient root colonization of plant growth-promoting rhizobacteria is critical for their plant-beneficial functions. However, the strategy to overcome plant immunity during root colonization is not well understood. In particular, how Bacillus strains cope with plant-derived reactive oxygen species (ROS), which function as the first barrier of plant defence, is not clear. In the present study, we found that the homolog of flg22 in Bacillus velezensis SQR9 (flg22 SQR9 ) has 78.95% identity to the typical flg22 (flg22 P.s. ) and induces a significant oxidative burst in cucumber and Arabidopsis. In contrast to pathogenic or beneficial Pseudomonas, live B. velezensis SQR9 also induced an oxidative burst in cucumber. We further found that B. velezensis SQR9 tolerated higher H 2 O 2 levels than Pst DC3000, the pathogen that harbours the typical flg22, and that it possesses the ability to suppress the flg22-induced oxidative burst, indicating that B. velezensis SQR9 may exploit a more efficient ROS tolerance system than DC3000. Further experimentation with mutagenesis of bacteria and Arabidopsis showed that the two-component regulatory system, ResDE, in B. velezensis SQR9 is involved in tolerance to plant-derived oxidative stress, thus contributing to root colonization. This study supports a further investigation of the interaction between beneficial rhizobacteria and plant immunity., (© 2021 John Wiley & Sons Ltd.)

Interbacterial antagonism and communication are driving forces behind microbial community development. In many Gram-negative bacteria, contact-dependent growth inhibition (CDI) systems contribute to these microbial interactions. CDI systems deliver the toxic C-terminus of a large surface exposed protein to the cytoplasm of neighboring bacteria upon cell-contact. Termed the BcpA-CT, import of this toxic effector domain is mediated by specific, yet largely unknown receptors on the recipient cell outer and inner membranes. In this study, we demonstrated that cytoplasmic membrane proteins GltJK, components of a predicted ABC-type transporter, are required for entry of CDI system protein BcpA-2 into Burkholderia multivorans recipient cells. Consistent with current CDI models, gltJK were also required for recipient cell susceptibility to a distinct BcpA-CT that shared sequences within the predicted "translocation domain" of BcpA-2. Strikingly, this translocation domain showed low sequence identity to the analogous region of an Escherichia coli GltJK-utilizing CDI system protein. Our results demonstrated that recipient bacteria expressing E. coli gltJK were resistant to BcpA-2-mediated interbacterial antagonism, suggesting that BcpA-2 specifically recognizes Burkholderia GltJK. Using a series of chimeric proteins, the specificity determinant was mapped to Burkholderia-specific sequences at the GltK C-terminus, providing insight into BcpA transport across the recipient cell cytoplasmic membrane., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Temperature sensing is a ubiquitous cell behavior, but the fundamental limits to the precision of temperature sensing are poorly understood. Unlike in chemical concentration sensing, the precision of temperature sensing is not limited by extrinsic fluctuations in the temperature field itself. Instead, we find that precision is limited by the intrinsic copy number, turnover, and binding kinetics of temperature-sensitive proteins. Developing a model based on the canonical TlpA protein, we find that a cell can estimate temperature to within 2%. We compare this prediction with in vivo data on temperature sensing in bacteria.

BceF is a bacterial tyrosine kinase (BY-kinase) from Burkholderia cepacia , a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients. BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections. BY-kinases share no homology with mammalian kinases, and thereby offer a means to develop novel and specific antivirulence drugs. Here, we report the crystal structure of the BceF kinase domain at 1.85 Å resolution. The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at the active sites. The low enzymatic efficiency measured in solution may be explained by the partial obstruction of the active sites at the crystallographic dimer interface. This study provides insights into self-assembly and the specific activity of isolated catalytic domains. Several unique variations around the active site compared to other BY-kinases may allow for structure-based design of specific inhibitors to target Burkholderia cepacia virulence.

Salmonella outer protein D (SopD) is secreted into a host during the first stages of the Salmonella infection and contributes to the systemic virulence of the bacterium. SopD2 is a SopD homolog and possesses GTPase activating protein (GAP) activity towards Rab32. Here, we identified Rab-proteins as putative SopD-targets using a yeast two-hybrid approach. In vitro investigations subsequently revealed Rab8a as an exclusive SopD substrate in contrast to SopD2, which has a broader specificity targeting Rab29, Rab32 and Rab38 in vitro. Additionally, we determined the catalytic efficiencies of SopD and SopD2 towards their physiologically relevant substrates. Moreover, mutagenesis studies provided insights into possible key residues of the Rab-protein and the GAP involved in the conversion of active to inactive GTPase. In conclusion, we demonstrate that Salmonella SopD and SopD2 act as RabGAPs and can inactivate Rab signaling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The effects of the bcsE gene and BcsE protein on bacterial physiology and pathogenicity in SalmonellaTyphimurium and Salmonella Group C1 were investigated. It was observed that biofilm and pellicle formation did not occur in the bcsE gene mutants of wild-type strains. Besides, the 'rdar' (red, dry, rough) biofilm morphotype in wild-type strains changed significantly in the mutants. In terms of the bcsE gene, the swimming and swarming motility in mutant strains showed a dramatic increase compared to the wild-type strains. The Salmonella bcsE gene was cloned into Escherichia coli BL21, and the his-tagged protein produced in this strain was purified to obtain polyclonal antibodies in BALB/c mice. The antibodies were showed labeled antigen specificity to the BscE protein. As a result of immunization and systemic persistence tests carried out with BALB/c mice, BscE protein was determined to trigger high levels of humoral and cellular responses (Th1 cytokine production, IgG2a/IgG1 > 1). Systemic persistence in the liver and spleen samples decreased by 99.99% and 100% in the bcsE mutant strains. Finally, invasion abilities on HT-29 epithelial cells of wild-type strains were utterly disappeared in their bcsE gene mutant strains., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The placenta controls the growth of the fetus and ensures its immune protection. Key to these functions, the syncytiotrophoblast (SYN) is a syncytium formed by fusion of underlying mononuclear trophoblasts. The SYN covers the placental surface and is bathed in maternal blood to mediate nutritional and waste exchanges between the mother and fetus. The bacterial pathogen Listeria monocytogenes breaches the trophoblast barrier and infects the placental/fetal unit resulting in poor pregnancy outcomes. In this work, we analyzed the L. monocytogenes intracellular lifecycle in primary human trophoblasts. In accordance with previous studies, we found that the SYN is 20-fold more resistant to infection compared to mononuclear trophoblasts, forming a protective barrier to infection at the maternal interface. We show for the first time that this is due to a significant reduction in L. monocytogenes uptake by the SYN rather than inhibition of the bacterial intracellular division or motility. We here report the first transcriptomic analysis of L. monocytogenes -infected trophoblasts (RNA sequencing). Pathway analysis showed that infection upregulated TLR2, NOD-like, and cytosolic DNA sensing pathways, as well as downstream pro-inflammatory circuitry (NF-κB, AP-1, IRF4, IRF7) leading to the production of mediators known to elicit the recruitment and activation of maternal leukocytes (IL8, IL6, TNFα, MIP-1). Signature genes associated with poor pregnancy outcomes were also upregulated upon infection. Measuring the release of 54 inflammatory mediators confirmed the transcriptomic data and revealed sustained production of tolerogenic factors (IL-27, IL-10, IL-1RA, TSLP) despite infection. Both the SYN and mononuclear trophoblasts produced cytokines, but surprisingly, some cytokines were predominantly produced by the SYN (IL-8, IL-6) or by non-fused trophoblasts (TNFα). Collectively, our data support that trophoblasts act as placental gatekeepers that limit and detect L. monocytogenes infection resulting in a pro-inflammatory response, which may contribute to the poor pregnancy outcomes if the pathogen persists., 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 © 2021 Johnson, Azari, Webb, Zhang, Gavrilin, Marshall, Rood and Seveau.)

The basal body of the bacterial flagellum is a rotary motor that consists of several rings (C, MS and LP) and a rod. The LP ring acts as a bushing supporting the distal rod for its rapid and stable rotation without much friction. Here, we use electron cryomicroscopy to describe the LP ring structure around the rod, at 3.5 Å resolution, from Salmonella Typhimurium. The structure shows 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners, which explains the structural stability. The inner surface is charged both positively and negatively. Positive charges on the P ring (the part of the LP ring that is embedded within the peptidoglycan layer) presumably play important roles in its initial assembly around the rod with a negatively charged surface., (© 2021. The Author(s).)

Aim: Chlamydia trachomatis has evolved various strategies to alleviate oxidative stress of host cells to maintain their intracellular survival. However, the exact mechanism of anti-oxidative stress of C. trachomatis is still unclear. The activation of nuclear factor erythroid 2-related factor 2/quinone oxidoreductase (Nrf2/NQO1) signal pathway has been identified as an efficient antioxidant defensive mechanism used by host cells to counteract oxidative stress. Pgp3 is a pivotal virulence factor of C. trachomatis involved in intracellular survival. The aim of this study is to explore the role of Pgp3 on Nrf2/NQO1 signal pathway against oxidative stress., Main Methods: After HeLa cells were stimulated with Pgp3 protein, Nrf2 location and the inclusion bodies of C. trachomatis were detected by indirect immunofluorescence, western blotting and Oxidative stress assay kits were used to separately determine the protein expression and the content of malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) before and after the interference of Nrf-2 and NQO1., Key Findings: Pgp3 promoted the nuclear translocation of Nrf2 to increase NQO1 expression and reduced oxidative stress induced by LPS to contribute to the survival of C. trachomatis. Inhibition of Nrf2/NQO1 signal pathway with Nrf2 inhibitor and down-regulation of NQO1 with siRNA-NQO1 suppressed oxidative stress resistance induced by Pgp3., Significance: Here we found that Pgp3 alleviated oxidative stress to promote the infectivity of C. trachomatis through activation of Nrf2/NQO1 signal pathway, which provided a novel understanding of the effects of Pgp3 in the pathogenesis of C. trachomatis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Helicobacter pylori is a gram-negative, microaerophilic, pathogenic bacterium and a widespread colonizer of humans. H. pylori has developed mechanisms that enable it to overcome the harsh environment of the human stomach, including reactive oxygen species (ROS). Interestingly, up to now no typical regulator dedicated to the oxidative-stress response has been discovered. In this work, we reveal that the inhibitor of replication initiation HP1021 functions as a redox switch protein in H. pylori and plays an important role in response to oxidative stress of the gastric pathogen. Each of the two predicted HP1021 domains contains three cysteine residues. We show that the cysteine residues of HP1021 are sensitive to oxidation both in vitro and in vivo, and we demonstrate that HP1021 DNA-binding activity to oriC depends on the redox state of the protein. Moreover, Zn2+ modulates HP1021 affinity towards oriC template DNA. Transcription analysis of selected H. pylori genes by RT-qPCR indicated that HP1021 is directly involved in the oxygen-dependent control of H. pylori fecA3 and gluP genes, which are implicated in response to oxidative stress. In conclusion, HP1021 is a redox switch protein and could be a target for H. pylori control strategies., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Neisseria gonorrhoeae is a bacterial pathogen that colonizes mucosal epithelia that are rich in antimicrobial molecules such as long-chain fatty acids. Here we studied the mechanisms involved in palmitic acid resistance and their impact on in vivo biological fitness in a murine genital tract infection model. A stable palmitic acid-resistant derivative was obtained by serial passage with incremental palmitic acid concentrations. This derivative outcompeted its parent strain for colonization and survival in the murine infection model. Subsequent whole-genome sequencing resulted in the identification of the 3 resistance-related SNPs ihfAC5T, fadDC772T, and farAG-52T (promoter) that were verified for resistance against palmitic acid. Subsequent characterization of the associated resistance determinants showed that ihfAC5T and farAG-52T induced gene expression of the FarAB efflux pump, whereas fadDC772T increased the maximum enzyme activity of the FadD long-chain fatty acid-coenzyme A ligase. Our results highlight the mechanisms involved in gonococcal adaptation to the murine host environment., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Type IV pili (T4P) are functionally versatile filamentous nanomachines, nearly ubiquitous in prokaryotes. They are predominantly polymers of one major pilin but also contain minor pilins whose functions are often poorly defined and likely to be diverse. Here, we show that the minor pilin PilB from the T4P of Streptococcus sanguinis displays an unusual bimodular three-dimensional structure with a bulky von Willebrand factor A-like (vWA) module "grafted" onto a small pilin module via a short loop. Structural modeling suggests that PilB is only compatible with a localization at the tip of T4P. By performing a detailed functional analysis, we found that 1) the vWA module contains a canonical metal ion-dependent adhesion site, preferentially binding Mg 2+ and Mn 2+ , 2) abolishing metal binding has no impact on the structure of PilB or piliation, 3) metal binding is important for S. sanguinis T4P-mediated twitching motility and adhesion to eukaryotic cells, and 4) the vWA module shows an intrinsic binding ability to several host proteins. These findings reveal an elegant yet simple evolutionary tinkering strategy to increase T4P functional versatility by grafting a functional module onto a pilin for presentation by the filaments. This strategy appears to have been extensively used by bacteria, in which modular pilins are widespread and exhibit an astonishing variety of architectures., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Quorum-sensing (QS) signals are widely employed by bacteria to regulate biological functions in response to cell densities. Previous studies showed that Burkholderia cenocepacia mostly utilizes two types of QS systems, including the N -acylhomoserine lactone (AHL) and cis -2-dodecenoic acid (BDSF) systems, to regulate biological functions. We demonstrated here that a LysR family transcriptional regulator, Bcal3178, controls the QS-regulated phenotypes, including biofilm formation and protease production, in B. cenocepacia H111. Expression of Bcal3178 at the transcriptional level was obviously downregulated in both the AHL-deficient and BDSF-deficient mutant strains compared to the wild-type H111 strain. It was further identified that Bcal3178 regulated target gene expression by directly binding to the promoter DNA regions. We also revealed that Bcal3178 was directly controlled by the AHL system regulator CepR. These results show that Bcal3178 is a new downstream component of the QS signaling network that modulates a subset of genes and functions coregulated by the AHL and BDSF QS systems in B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is an important opportunistic pathogen in humans that utilizes the BDSF and AHL quorum-sensing (QS) systems to regulate biological functions and virulence. We demonstrated here that a new downstream regulator, Bcal3178 of the QS signaling network, controls biofilm formation and protease production. Bcal3178 is a LysR family transcriptional regulator modulated by both the BDSF and AHL QS systems. Furthermore, Bcal3178 controls many target genes, which are regulated by the QS systems in B. cenocepacia. Collectively, our findings depict a novel molecular mechanism with which QS systems regulate some target gene expression and biological functions by modulating the expression level of a LysR family transcriptional regulator in B. cenocepacia.

Background: Campylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates., Methods: A total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping., Results: Out of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates., Conclusion: Significant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

This review focuses on the molecular chaperone ClpB that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and its biological function in selected bacterial pathogens, causing a variety of human infectious diseases, including zoonoses. It has been established that ClpB disaggregates and reactivates aggregated cellular proteins. It has been postulated that ClpB's protein disaggregation activity supports the survival of pathogenic bacteria under host-induced stresses (e.g., high temperature and oxidative stress), which allows them to rapidly adapt to the human host and establish infection. Interestingly, ClpB may also perform other functions in pathogenic bacteria, which are required for their virulence. Since ClpB is not found in human cells, this chaperone emerges as an attractive target for novel antimicrobial therapies in combating bacterial infections.

Multidrug and toxic compound extrusion (MATE) transport proteins confer multidrug resistance on pathogenic microorganisms and affect pharmacokinetics in mammals. Our understanding of how MATE transporters work, has mostly relied on protein structures and MD simulations. However, the energetics of drug transport has not been studied in detail. Many MATE transporters utilise the electrochemical H + or Na + gradient to drive substrate efflux, but NorM-VC from Vibrio cholerae can utilise both forms of metabolic energy. To dissect the localisation and organisation of H + and Na + translocation pathways in NorM-VC we engineered chimaeric proteins in which the N-lobe of H + -coupled NorM-PS from Pseudomonas stutzeri is fused to the C-lobe of NorM-VC, and vice versa. Our findings in drug binding and transport experiments with chimaeric, mutant and wildtype transporters highlight the versatile nature of energy coupling in NorM-VC, which enables adaptation to fluctuating salinity levels in the natural habitat of V. cholerae.

Pseudomonas aeruginosa is an opportunistic pathogen, mainly affecting severe patients, such as those in intensive care units (ICUs). High levels of antibiotic resistance and a long battery of virulence factors characterise this pathogen. Among virulence factors, the T3SS (Type 3 Secretion Systems) are especially relevant, being one of the most important virulence factors in P. aeruginosa. T3SS are a complex "molecular syringe" able to inject different effectors in host cells, subverting cell machinery influencing immune responses, and increasing bacterial survival rates. While T3SS have been largely studied and the molecular structure and main effector functions have been established, a series of questions and further points remain to be clarified or established. The key role of T3SS in P. aeruginosa virulence has resulted in the search for T3SS-targeting molecules able to impair their functions and subsequently improve patient outcomes. This review aims to summarise the most relevant features of the P. aeruginosa T3SS., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis with millions of deaths annually, remains one of the most formidable pathogen to global public health. As the most successful intracellular pathogens, Mtb can spatiotemporally coordinate the transcription and translation timely to reconcile the inevitable transcription-replication conflicts. Mutation frequency decline (Mfd) is a bacterial ATP-dependent DNA translocase that couples DNA repair to transcription via hydrolyzing ATP as energy, which preferentially acts on the damaged DNA transcribed strand to rescue stalled RNAP or dissociate RNAP to terminate the transcription depending on impediment severity, mitigating the damage to bacteria. In addition to the traditional damage repair effect, Mfd may also promote bacteria mutagenesis under stresses and boost the drug resistance. Mfd is widespread among bacteria and intensively studied, but there are very few studies in Mycobacteria, especially Mtb. In this review, the structure, function and mechanism characteristics of Mfd in Mtb (MtbMfd, Rv1020) are explored, with emphasis on the regulatory network of MtbMfd and its potential as a prime target for antibiotic drugs against tuberculosis., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Edwardsiella piscicida (E. piscicida) is an important fish pathogen. However, the mechanism of Glu6P transport regulatory protein UhpA how to affect the virulence gene expression in E. piscicida is still unclear. The results in this study showed that the metabolism-related gene expression of cysteine synthase (orf 1134) and sulphate transporter (ychM) in the uhpA mutant strain ΔuhpA was 0.76-fold and 0.68-fold lower than the ones in the wild strains (p < .05). The gene expression of ethA and ethB in the ΔuhpA strain was 0.80-fold and 0.72-fold lower than the ones in the wild strains (p < .05). However, the gene expression of fliC and flgN in the ΔuhpA was 1.51-fold and 1.21-fold higher than the ones in the wild strains (p < .05). The gene expression of T3SS (esrB and esrC) and T6SS (evpB and evpC) in the ΔuhpA was 1.27-fold, 1.13-fold, 1.28-fold and 1.23-fold higher than the ones in the wild strains (p < .05). This suggested that the uhpA gene could regulate the key virulence gene expression, and the uhpA gene was associated with the pathogenicity of E. piscicida in fish., (© 2020 John Wiley & Sons Ltd.)