Type III Secretion Research Articles

Primary Infection and Supernatants Generated by Pseudomonas aeruginosa ExoY Have Lasting Effects on Cyclic Nucleotide Signaling in Pulmonary Endothelial Cells

Rationale: Pseudomonas aeruginosa is an opportunistic pathogen and a common cause of hospital-acquire pneumonia. This gram-negative bacterium has an arsenal of virulence factors including the type three secretion system (TTSS), which encodes for an injection type needle and exotoxins. One of the four exotoxins, ExoY, is a promiscuous nucleotidylyl cyclase that utilizes F-actin as a cofactor to generate both canonical and non-canonical cyclic nucleotides (cAMP, cGMP and cCMP and cUMP respectively). While it is well known that activation of endogenous adenylyl and guanylyl cyclase leads to intracellular increases in cAMP and cGMP and release into the extracellular space, whether ExoY generated cyclic nucleotides are also effluxed out of the cell into the extracellular space is not known. In addition, it is unclear whether presence of the bacteria themselves affects endogenous cAMP signaling. Further, infection with P. aeruginosa expressing ExoY generates virulent cytotoxic amyloids from lung endothelium; however, whether these cytotoxic amyloids disrupt agonist-induced endogenous cAMP generation is unknown. Thus, we tested the hypothesis that during primary infection, cAMP and cGMP are effluxed into the extracellular space, while the primary infection itself and sterile supernatants from the primary infection attenuate isoproterenol induce increases in cAMP in pulmonary microvascular endothelial cells (PMVECs). Methods: During primary infection, PMVECs were inoculated with PBS (vehicle control) or 3 different strains of P. aeruginosa at an moi 20:1. Strains ExoY+ and ExoYK81M have a fully functional TTSS and express either catalytically active or inactive ExoY, respectively, while a third strain, ExoYTM, is unable to translocate ExoY into eukaryotic cells. During the last 15 minutes of the 4-hour infection, cells were stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. In separate experiments, supernatants from a primary infection were generated using the same three strains of bacteria at moi 20:1 and 4 hours after inoculation, the supernatants collected and sterilized using a 0.2 μm filter. Sterilized supernatants were added to naïve PMVECs and during the last 15 minutes of the 4-hour inoculation, cells stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. Results: Cyclic AMP and cGMP were elevated in both the lysate and media of PMVECs following 4-hour infection with ExoY+ and the total isoproterenol and rolipram-stimulated cAMP response was attenuated by the presence of bacteria, ExoYK81M and ExoYTM, and further blunted with ExoY+ infection. Further, inoculation with ExoYK81M and ExoYTM supernatants attenuated the total isoproterenol and rolipram-stimulated cAMP response, which was further blunted by ExoY+ supernatants. Conclusions: Overall, we demonstrate PMVECs release a large cAMP and cGMP pool into the extracellular space and both primary infection and supernatants derived from the primary infection attenuate agonist induced elevations in cAMP. HL121513, HL66299. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AAV-vectored expression of monospecific or bispecific monoclonal antibodies protects mice from lethal Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa poses a significant threat to immunocompromised individuals and those with cystic fibrosis. Treatment relies on antibiotics, but persistent infections occur due to intrinsic and acquired resistance of P. aeruginosa towards multiple classes of antibiotics. To date, there are no licensed vaccines for this pathogen, prompting the urgent need for novel treatment approaches to combat P. aeruginosa infection and persistence. Here we validated AAV vectored immunoprophylaxis as a strategy to generate long-term plasma and mucosal expression of highly protective monoclonal antibodies (mAbs) targeting the exopolysaccharide Psl (Cam-003) and the PcrV (V2L2MD) component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. When administered intramuscularly, AAV-αPcrV, AAV-αPsl, and AAV-MEDI3902 significantly protected mice challenged intranasally with a lethal dose of P. aeruginosa strains PAO1 and PA14 and reduced bacterial burden and dissemination to other organs. While all AAV-mAbs provided protection, AAV-αPcrV and AAV-MEDI3902 provided 100% and 87.5% protection from a lethal challenge with 4.47 × 107 CFU PAO1 and 87.5% and 75% protection from a lethal challenge with 3 × 107 CFU PA14, respectively. Serum concentrations of MEDI3902 were ~10× lower than that of αPcrV, but mice treated with this vector showed a greater reduction in bacterial dissemination to the liver, lung, spleen, and blood compared to other AAV-mAbs. These results support further investigation into the use of AAV vectored immunoprophylaxis to prevent and treat P. aeruginosa infections and other bacterial pathogens of public health concern for which current treatment strategies are limited.

Chromosomal and plasmid-encoded virulence and multidrug resistance of Escherichia coli ST58/24 infecting a 2-year-old sickle cell patient with sepsis in Kampala Uganda, East Africa

Sepsis and drug resistance represent a complex of the most common global causes of mortality in intensive care units (ICUs) especially among patients with comorbidities. Extraintestinal pathogenic Escherichia coli (ExPEC) strains are highly implicated in systemic infections, with multidrug resistance exacerbating the risk of chronic conditions and patient mortality. The diversity of virulence and evolution of multidrug resistance are yet to be fully deciphered. In this work, we aimed at unveiling the pathogens and their genomic determinants of virulence and drug resistance relevant to increased sepsis in a sickle cell child admitted to ICU. From a rectal swab, we isolated a strain of E. coli from the patient and phenotypically tested it against a panel of selected beta lactams, fluoroquinolones, macrolides, aminoglycosides and colistin. We then sequenced the entire genome and integrated multiple bioinformatic pipelines to divulge the virulence and multidrug resistance profiles of the isolate. Our results revealed that the isolate belongs to the sequence type (ST) 58/24, which (ST58), is a known ExPEC. With the use of PathogenFinder, we were able to confirm that this isolate is a human pathogen (p = 0.936). The assembled chromosome and two plasmids encode virulence factors related to capsule (antiphagocytosis), serum survival and resistance, type 6 secretion system (T6SS), multiple siderophores (iron acquisition), and biosynthetic gene clusters for polyketides and nonribosomal peptides exhibiting host cell damaging activity in silico. The genome also harbors multidrug resistance genotypes including extended spectrum beta lactamase (ESBL) genes such as blaTEM-1A/B, sulfonamide resistance genes sul1/2, fluoroquinolone resistance genes dfrA5 and nonsynonymous mutations of the gene pmrB, conferring intrinsic colistin resistance. Conclusively, this pathogen holds the potential to cause systemic infection and might exacerbate sickle cell anemia in the patient. The virulence and multidrug resistance profiles are encoded by both the chromosome and plasmids. Genomic surveillance of pathogens with multidrug resistance among patients with comorbidities is crucial for effective disease management.

Plant-Derived Phenolic Acids Limit the Pathogenesis of Salmonella Typhimurium and Protect Intestinal Epithelial Cells during Their Interactions.

The incidence of gastrointestinal illness attributable to Salmonella enterica serovar Typhimurium (ST) remains a concern for public health worldwide, as it can progress into systemic infections mediated by the type-three secretion system (T3SS), which allows for adherence and invasion to intestinal epithelial cells. The current study evaluates the ability of gallic acid (GA), protocatechuic acid (PA), and vanillic acid (VA) to impair the adhesion and invasion abilities of ST to a human epithelial (INT-407) cell monolayer while also assessing their cytotoxicity. GA, PA, and VA inhibited detectable ST growth at specific concentrations but showed cytotoxicity against INT-407 cells (>20% reduction in viability) after 3 h of treatments. Adjusting the pH of the solutions had a neutralizing effect on cytotoxicity, though it did reduce their antimicrobial potency. Adhesion of ST was reduced significantly when the cells were treated with 4.0 mg/mL of VA, whereas invasion was reduced in all treatments, with GA requiring the lowest concentration (0.5 mg/mL). Relative gene expression of virulence genes after treatment with GA showed downregulation in the T3SS regulator and effector hilA and sipA, respectively. These findings suggest further use of phenolic acids in reducing the activity of key virulence factors critical during ST infection.

CobB-mediated deacetylation of the chaperone CesA regulates Escherichia coli O157:H7 virulence

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a common food-borne pathogen that can cause acute diseases. Lysine acetylation is a post-translational modification (PTM) that occurs in various prokaryotes and is regulated by CobB, the only deacetylase found in bacteria. Here, we demonstrated that CobB plays an important role in the virulence of EHEC O157:H7 and that deletion of cobB significantly decreased the intestinal colonization ability of bacteria. Using acetylation proteomic studies, we systematically identified several proteins that could be regulated by CobB in EHEC O157:H7. Among these CobB substrates, we found that acetylation at the K44 site of CesA, a chaperone for the type-III secretion system (T3SS) translocator protein EspA, weakens its binding to EspA, thereby reducing the stability of this virulence factor; this PTM ultimately attenuating the virulence of EHEC O157:H7. Furthermore, we showed that deacetylation of the K44 site, which is deacetylated by CobB, promotes the interaction between CesA and EspA, thereby increasing bacterial virulence in vitro and in animal experiments. In summary, we showed that acetylation influences the virulence of EHEC O157:H7, and uncovered the mechanism by which CobB contributes to bacterial virulence based on the regulation of CesA deacetylation.

Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks.

Bacterial competition may rely on secretion systems such as the type 6 secretion system (T6SS), which punctures and releases toxic molecules into neighboring cells. To subsist, bacterial targets must counteract the threats posed by T6SS-positive competitors. In this study, we used a comprehensive genome-wide high-throughput screening approach to investigate the dynamics of interbacterial competition. Our primary goal was to identify deletion mutants within the well-characterized E. coli K-12 single-gene deletion library, the Keio collection, that demonstrated resistance to T6SS-mediated killing by the enteropathogenic bacterium Cronobacter malonaticus. We identified 49 potential mutants conferring resistance to T6SS and focused our interest on a deletion mutant (∆fimE) exhibiting enhanced expression of type 1 fimbriae. We demonstrated that the presence of type 1 fimbriae leads to the formation of microcolonies and thus protects against T6SS-mediated assaults. Collectively, our study demonstrated that adhesive structures such as type 1 fimbriae confer collective protective behavior against T6SS attacks.IMPORTANCEType 6 secretion systems (T6SS) are molecular weapons employed by gram-negative bacteria to eliminate neighboring microbes. T6SS plays a pivotal role as a virulence factor, enabling pathogenic gram-negative bacteria to compete with the established communities to colonize hosts and induce infections. Gaining a deeper understanding of bacterial interactions will allow the development of strategies to control the action of systems such as the T6SS that can manipulate bacterial communities. In this context, we demonstrate that bacteria targeted by T6SS attacks from the enteric pathogen Cronobacter malonaticus, which poses a significant threat to infants, can develop a collective protective mechanism centered on the production of type I fimbriae. These adhesive structures promote the aggregation of bacterial preys and the formation of microcolonies, which protect the cells from T6SS attacks.

ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis.

Salmonella enterica is a major pathogen responsible for foodborne gastroenteritis, and a leading model organism for genetic and molecular studies of bacterial virulence mechanisms. One key trait of this pathogen is the ability to survive within infected host cells. During infection, the bacteria employ a type three secretion system that deliver effector proteins to target and manipulate host cell processes. The transcriptional regulation of this virulence program is well understood. By contrast, the factors and mechanisms operating at the post-transcriptional level to control virulence gene expression are less clear. In this study, we have charted the global RNA ligand repertoire of the RNA-binding protein ProQ during in vitro conditions mimicking the host cell environment. This identified hundreds of binding sites and revealed ProQ-dependent stabilization of intracellular-specific small RNAs. Importantly, we show that ProQ post-transcriptionally activates the expression of PhoP, a master transcriptional activator of intracellular virulence in Salmonella.

Type 3 secretion system induced leukotriene B4 synthesis by leukocytes is actively inhibited by Yersinia pestis to evade early immune recognition.

Subverting the host immune response to inhibit inflammation is a key virulence strategy of Yersinia pestis. The inflammatory cascade is tightly controlled via the sequential action of lipid and protein mediators of inflammation. Because delayed inflammation is essential for Y. pestis to cause lethal infection, defining the Y. pestis mechanisms to manipulate the inflammatory cascade is necessary to understand this pathogen's virulence. While previous studies have established that Y. pestis actively inhibits the expression of host proteins that mediate inflammation, there is currently a gap in our understanding of the inflammatory lipid mediator response during plague. Here we used the murine model to define the kinetics of the synthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid chemoattractant and immune cell activator, within the lungs during pneumonic plague. Furthermore, we demonstrated that exogenous administration of LTB4 prior to infection limited bacterial proliferation, suggesting that the absence of LTB4 synthesis during plague contributes to Y. pestis immune evasion. Using primary leukocytes from mice and humans further revealed that Y. pestis actively inhibits the synthesis of LTB4. Finally, using Y. pestis mutants in the Ysc type 3 secretion system (T3SS) and Yersinia outer protein (Yop) effectors, we demonstrate that leukocytes recognize the T3SS to initiate the rapid synthesis of LTB4. However, several Yop effectors secreted through the T3SS effectively inhibit this host response. Together, these data demonstrate that Y. pestis actively inhibits the synthesis of the inflammatory lipid LTB4 contributing to the delay in the inflammatory cascade required for rapid recruitment of leukocytes to sites of infection.

Regulation of virulence in Chromobacterium violaceum and strategies to combat it.

Chromobacterium is a rod-shaped, Gram-negative, facultatively anaerobic bacteria with a cosmopolitan distribution. Just about 160 Chromobacterium violaceum incidents have been reported globally, but then once infected, it has the ability to cause deadly septicemia, and infections in the lungs, liver, brain, spleen, and lymphatic systems that might lead to death. C. violaceum produces and utilizes violacein to kill bacteria that compete with it in an ecological niche. Violacein is a hydrophobic bisindole that is delivered through an efficient transport route termed outer membrane vesicles (OMVs) through the aqueous environment. OMVs are small, spherical segments detached from the outer membrane of Gram-negative bacteria. C. violaceum OMV secretions are controlled by a mechanism called the quorum sensing system CviI/CviR, which enables cell-to-cell communication between them and regulation of various virulence factors such as biofilm formation, and violacein biosynthesis. Another virulence factor bacterial type 3 secretion system (T3SS) is divided into two types: Cpi-1 and Cpi-2. Cpi-1's needle and rod effector proteins are perhaps recognized by NAIP receptors in humans and mice, activating the NLRC4 inflammasome cascade, effectively clearing spleen infections via pyroptosis, and cytotoxicity mediated by IL-18-driven Natural killer (NK) cells in the liver. In this paper, we attempt to interrelate quorum-controlled biofilm formation, violacein production, violacein delivery by OMVs and T3SS effector protein production and host mediated immunological effects against the Cpi1 of T3SS. We suggest a research path with natural bioactive molecule like palmitic acid that can act as an anti-quorum agent by reducing the expression of virulence factors as well as an immunomodulatory agent that can augment innate immune defense by hyperactivation of NLRC4 inflammasome hence dramatically purge C. violaceum infections.

Isolation, identification and characterization of Aeromonas jandaei from diseased Chinese soft-shell turtles.

Aeromonas jandaei is a gram-negative bacterium commonly found in aquatic environments and can induce illnesses in amphibians, reptiles and aquatic animals. In this study, a strain of bacteria was isolated from the diseased Chinese soft-shell turtle (Pelodiscus sinensis), then named strain JDP-FX. This isolate was identified as A. jandaei after analysis of morphological, physiological and biochemical characteristics, as well as 16S rRNA and gyrB gene sequences. Virulence genetic testing further detected temperature-sensitive protease (eprCAI), type III secretion system (TTSS) (ascv), nuclease (nuc), cytotonic enterotoxin (alt) and serine proteinase (ser) in JDP-FX. Compared with healthy Chinese soft-shell turtle, the serum levels of total protein (TP), albumin (ALB) and globulin (GLB) were significantly decreased in the diseased Chinese soft-shell turtle, while, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly increased. Histopathological observations showed that multiple tissues, including intestinal mucosa, liver and kidney, were severely damaged in the diseased Chinese soft-shell turtle. Moreover, the diseased Chinese soft-shell turtle had significant cell degeneration, necrosis, sloughing and interstitial inflammatory cell infiltration. The pathogenicity of JDP-FX was tested via artificial infection. The median lethal dosage (LD50 ) of the strain was 1.05 × 105 colony forming units (CFU/g) per weight of Chinese soft-shell turtle. Drug susceptibility analysis revealed that JDP-FX was susceptible to ceftazidime, minocycline, cefoperazone, ceftriaxone and piperacillin. In addition, JDP-FX was resistant to doxycycline, florfenicol, sulfonamides, gentamicin, ampicillin and neomycin. Therefore, this study may provide guidance for further research into the diagnosis, prevention and treatment of JDP-FX infection.

Structure and Dynamics of Type 4a Pili and Type 2 Secretion System Endopili.

Within the highly diverse type four filament (TFF or T4F) superfamily, the machineries of type IVa pili (T4aP) and the type 2 secretion system (T2SS) in diderm bacteria exhibit a substantial sequence similarity despite divergent functions and distinct appearances: T4aP can extend micrometers beyond the outer membrane, whereas the endopili in the T2SS are restricted to the periplasm. The determination of the structure of individual components and entire filaments is crucial to understand how their structure enables them to serve different functions. However, the dynamics of these filaments poses a challenge for their high-resolution structure determination. This review presents different approaches that have been used to study the structure and dynamics of T4aP and T2SS endopili by means of integrative structural biology, cryo-electron microscopy (cryo-EM), and molecular dynamics simulations. Their conserved features and differences are presented. The non-helical stretch in the long-conserved N-terminal helix which is characteristic of all members of the TFF and the impact of calcium on structure, function, and dynamics of these filaments are discussed in detail.

Leaf microbiome dysbiosis triggered by T2SS-dependent enzyme secretion from opportunistic Xanthomonas pathogens

In healthy plants, the innate immune system contributes to maintenance of microbiota homoeostasis, while disease can be associated with microbiome perturbation or dysbiosis, and enrichment of opportunistic plant pathogens like Xanthomonas. It is currently unclear whether the microbiota change occurs independently of the opportunistic pathogens or is caused by the latter. Here we tested if protein export through the type-2 secretion system (T2SS) by Xanthomonas causes microbiome dysbiosis in Arabidopsis thaliana in immunocompromised plants. We found that Xanthomonas strains secrete a cocktail of plant cell wall-degrading enzymes that promote Xanthomonas growth during infection. Disease severity and leaf tissue degradation were increased in A. thaliana mutants lacking the NADPH oxidase RBOHD. Experiments with gnotobiotic plants, synthetic bacterial communities and wild-type or T2SS-mutant Xanthomonas revealed that virulence and leaf microbiome composition are controlled by the T2SS. Overall, a compromised immune system in plants can enrich opportunistic pathogens, which damage leaf tissues and ultimately cause microbiome dysbiosis by facilitating growth of specific commensal bacteria.

Genes associated with fitness and disease severity in the pan-genome of mastitis-associated Escherichia coli.

Bovine mastitis caused by Escherichia coli compromises animal health and inflicts substantial product losses in dairy farming. It may manifest as subclinical through severe acute disease and can be transient or persistent in nature. Little is known about bacterial factors that impact clinical outcomes or allow some strains to outcompete others in the mammary gland (MG) environment. Mastitis-associated E. coli (MAEC) may have distinctive characteristics which may contribute to the varied nature of the disease. Given their high levels of intraspecies genetic variability, virulence factors of commonly used MAEC model strains may not be relevant to all members of this group. In this study, we sequenced the genomes of 96 MAEC strains isolated from cattle with clinical mastitis (CM). We utilized clinical severity data to perform genome-wide association studies to identify accessory genes associated with strains isolated from mild or severe CM, or with high or low competitive fitness during in vivo competition assays. Genes associated with mastitis pathogens or commensal strains isolated from bovine sources were also identified. A type-2 secretion system (T2SS) and a chitinase (ChiA) exported by this system were strongly associated with pathogenic isolates compared with commensal strains. Deletion of chiA from MAEC isolates decreased their adherence to cultured bovine mammary epithelial cells. The increased fitness associated with strains possessing this gene may be due to better attachment in the MG. Overall, these results provide a much richer understanding of MAEC and suggest bacterial processes that may underlie the clinical diversity associated with mastitis and their adaptation to this unique environment.

Stripe Rust Effector Pst_9302 Inhibits Wheat Immunity to Promote Susceptibility.

Puccinia striiformis f. sp. tritici is an obligate biotrophic fungus that causes destructive stripe rust disease in wheat. During infection, Pst secretes virulence effectors via a specific infection structure-the haustorium-inside host cells to disturb host immunity and promote fungal colonization and expansion. Hence, the identification and functional analyses of Pst effectors are of great significance in deciphering the Pst pathogenicity mechanism. Here, we identified one candidate Pst effector Pst_9302 that could suppress Bax-triggered cell death in Nicotiana benthamiana. qRT-PCR analyses showed that the transcript levels of Pst_9302 were highly increased during the early infection stages of Pst. The transient expression of Pst_9302 in wheat via the type-three secretion system (T3SS) significantly inhibited the callose deposition induced by Pseudomonas syringae EtHAn. During wheat-Pst interaction, Pst_9302 overexpression suppressed reactive oxygen species (ROS) accumulation and cell death caused by the avirulent Pst race CYR23. The host-induced gene silencing (HIGS) of Pst_9302 resulted in decreased Pst pathogenicity with reduced infection area. The results suggest that Pst_9302 plays a virulence role in suppressing plant immunity and promoting Pst pathogenicity. Moreover, wheat voltage-dependent anion channel 1 protein (TaVDAC1) was identified as candidate Pst_9302-interacting proteins by yeast two-hybrid (Y2H) screening. Pull-down assays using the His-Pst_9302 and GST-TaVDAC1 protein verified their interactions. These results suggest that Pst_9302 may modulate wheat TaVDAC1 to regulate plant immunity.

Gene expression changes throughout the life cycle allow a bacterial plant pathogen to persist in diverse environmental habitats.

Bacterial pathogens exhibit a remarkable ability to persist and thrive in diverse ecological niches. Understanding the mechanisms enabling their transition between habitats is crucial to control dissemination and potential disease outbreaks. Here, we use Ralstonia solanacearum, the causing agent of the bacterial wilt disease, as a model to investigate pathogen adaptation to water and soil, two environments that act as bacterial reservoirs, and compare this information with gene expression in planta. Gene expression in water resembled that observed during late xylem colonization, with an intriguing induction of the type 3 secretion system (T3SS). Alkaline pH and nutrient scarcity-conditions also encountered during late infection stages-were identified as the triggers for this T3SS induction. In the soil environment, R. solanacearum upregulated stress-responses and genes for the use of alternate carbon sources, such as phenylacetate catabolism and the glyoxylate cycle, and downregulated virulence-associated genes. We proved through gain- and loss-of-function experiments that genes associated with the oxidative stress response, such as the regulator OxyR and the catalase KatG, are key for bacterial survival in soil, as their deletion cause a decrease in culturability associated with a premature induction of the viable but non culturable state (VBNC). This work identifies essential factors necessary for R. solanacearum to complete its life cycle and is the first comprehensive gene expression analysis in all environments occupied by a bacterial plant pathogen, providing valuable insights into its biology and adaptation to unexplored habitats.

A growing battlefield in the war against biofilm-induced antimicrobial resistance: insights from reviews on antibiotic resistance.

Biofilms are a common survival strategy employed by bacteria in healthcare settings, which enhances their resistance to antimicrobial and biocidal agents making infections difficult to treat. Mechanisms of biofilm-induced antimicrobial resistance involve reduced penetration of antimicrobial agents, increased expression of efflux pumps, altered microbial physiology, and genetic changes in the bacterial population. Factors contributing to the formation of biofilms include nutrient availability, temperature, pH, surface properties, and microbial interactions. Biofilm-associated infections can have serious consequences for patient outcomes, and standard antimicrobial therapies are often ineffective against biofilm-associated bacteria, making diagnosis and treatment challenging. Novel strategies, including antibiotics combination therapies (such as daptomycin and vancomycin, colistin and azithromycin), biofilm-targeted agents (such as small molecules (LP3134, LP3145, LP4010, LP1062) target c-di-GMP), and immunomodulatory therapies (such as the anti-PcrV IgY antibodies which target Type IIIsecretion system), are being developed to combat biofilm-induced antimicrobial resistance. A multifaceted approach to diagnosis, treatment, and prevention is necessary to address this emerging problem in healthcare settings.

Cell-Based Assay to Determine Type 3 Secretion System Translocon Assembly in Pseudomonas aeruginosa Using Split Luciferase.

Multi-drug-resistant Pseudomonas aeruginosa poses a serious threat to hospitalized patients. This organism expresses an arsenal of virulence factors that enables it to readily establish infections and disseminate in the host. The Type 3 secretion system (T3SS) and its associated effectors play a crucial role in the pathogenesis of P. aeruginosa, making them attractive targets for the development of novel therapeutic agents. The T3SS translocon, composed of PopD and PopB, is an essential component of the T3SS secretion apparatus. In the properly assembled translocon, the N-terminus of PopD protrudes into the cytoplasm of the target mammalian cell, which can be exploited as a molecular indicator of functional translocon assembly. In this article, we describe a novel whole-cell-based assay that employs the split NanoLuc luciferase detection system to provide a readout for translocon assembly. The assay demonstrates a favorable signal/noise ratio (13.6) and robustness (Z' = 0.67), making it highly suitable for high-throughput screening of small-molecule inhibitors targeting T3SS translocon assembly.

Transcriptomic and phenotype analysis revealed the role of rpoS in stress resistance and virulence of a novel ST3355 ESBL-producing hypervirulent Klebsiella pneumoniae isolate.

An extended-spectrum beta-lactamase (ESBL)-hypervirulent Klebsiella pneumoniae (HvKP) strain HKE9 was isolated from the blood in an outpatient. The effect of the global regulatory factor RpoS on antimicrobial resistance, pathogenicity, and environmental adaptability was elucidated. HKE9 is a novel ST3355 (K20/O2a) hypervirulent strain with a positive string test and resistant to cephems except cefotetan. It has a genome size of 5.6M, including two plasmids. CTX-M-15 was found in plasmid 2, and only ompk37 was found in the chromosome. HKE9 could produce bacterial siderophores, and genes of enterobactin, yersiniabactin, aerobactin, and salmochelin have been retrieved in the genome. As a global regulatory factor, knockout of rpoS did not change antimicrobial resistance or hemolytic phenotype while increasing the virulence to Galleria mellonella larvae and showing higher viscosity. Moreover, rpoS knockout can increase bacterial competitiveness and cell adhesion ability. Interestingly, HKE9-M-rpoS decreased resistance to acidic pH, high osmotic pressure, heat shock, and ultraviolet and became sensitive to disinfectants (H2O2, alcohol, and sodium hypochlorite). Although there were 13 Type 6 secretion system (T6SS) core genes divided into two segments with tle1 between segments in the chromosome, transcriptomic analysis showed that rpoS negatively regulated T4SS located on plasmid 2, type 1, and type 3 fimbriae and positively regulate genes responsible for acidic response, hyperosmotic pressure, heat shock, oxidative stress, alcohol and hypochlorous acid metabolism, and quorum sensing. Here, this novel ST3355 ESBL-HvKP strain HKE9 may spread via various clonal types. The important regulation effect of rpoS is the enhanced tolerance and resistance to environmental stress and disinfectants, which may be at the cost of reducing virulence and regulated by T4SS.

L-glutamine protects against enterohemorrhagic Escherichia coli infection by inhibiting bacterial virulence and enhancing host defense concurrently.

Enterohemorrhagic Escherichia coli (EHEC) can colonize the gastrointestinal tract and cause bloody diarrhea in children. Previous studies showed that its pathogenesis could be mediated by metabolites from both the host and microbiota. L-Glutamine (Gln) was found to be depleted in intestinal tissues as the main energy source according to previous studies. Hence, we aimed to determine the effects of Gln on EHEC infection and its underlying mode of action. In this study, a Gln-limited signal was found to activate the type 3 secretion system (T3SS), which is crucial for EHEC infection via perturbation of central metabolism. By shifting the phosphorylation of NtrC, a key regulator in bacterial nitrogen metabolism, Gln stimulates ler transcripts in a σS-PchA-dependent manner. Our in vivo experiments further demonstrated that Gln supplementation can reduce EHEC colonization in the gastrointestinal tract by repressing T3SS. Moreover, Gln could further attenuate bacterial infection by boosting host defense, which might be dependent on multiple pathways. Besides, our experiments demonstrated that Gln did not induce Shiga-like toxin (Stx) production or cause impairment of gut flora. In conclusion, our study presented evidence that Gln could act against EHEC infection by reducing bacterial virulence and strengthening host defense. Therefore, Gln serves as a promising therapeutic agent for EHEC infection. IMPORTANCE The type 3 secretion system (T3SS) was obtained in many Gram-negative bacterial pathogens, and it is crucial for their pathogenesis. Environmental signals were found to be involved in the expression regulation of T3SS, which was vital for successful bacterial infection in the host. Here, we discovered that L-glutamine (Gln), the most abundant amino acid in the human body, could repress enterohemorrhagic Escherichia coli (EHEC) T3SS expression via nitrogen metabolism and therefore had potential as an antivirulence agent. Our in vitro and in vivo evidence demonstrated that Gln could decline EHEC infection by attenuating bacterial virulence and enhancing host defense simultaneously. We repurpose Gln as a potential treatment for EHEC infection accordingly.

Escherichia coli killing by epidemiologically successful sublineages of Shigella sonnei is mediated by colicins

Shigella sp. are enteric pathogens which causes >125 million cases of shigellosis annually. S.sonnei accounts for about a quarter of those cases and is increasingly prevalent in industrialising nations. Being an enteric pathogen, S.sonnei benefits from outcompeting gut commensals such as Escherichia coli to establish itself and cause disease. There are numerous mechanisms that bacterial pathogens use to outcompete its rivals including molecules called colicins. A Type 6 Secretion System (T6SS) was recently described as contributing to E.coli killing in S.sonnei. We used Bulk Phenotyping of Epidemiological Replicates (BPER) which combined bacterial Genome Wide Association Studies (bGWAS) and high throughput phenotyping on a collection of S.sonnei surveillance isolates to identify the genetic features associated with E.coli killing and explore their relationship with epidemiological behaviour. We further explored the presence of colicins and T6SS components in the isolates using genomics, laboratory experimentation, and proteomics. Our bGWAS analysis returned known and novel colicin and colicin related genes as significantly associated with E.coli killing. In silico analyses identified key colicin clusters responsible for the killing phenotype associated with epidemiologically successful sub-lineages. The killing phenotype was not associated with the presence of a T6SS. Laboratory analyses confirmed the presence of the key colicin clusters and that killing was contact-independent. Colicins are responsible for E.coli killing by S.sonnei, not a T6SS. This phenotype contributes to shaping the observed epidemiology of S.sonnei and may contribute to its increasing prevalence globally. BPER is an epidemiologically relevant approach to phenotypic testing that enables the rapid identification of genetic drivers of phenotypic changes, and assessment of their relevance to epidemiology in natural settings. Biotechnology and Biological Sciences Research Council, Biotechnology and Biological Sciences Research Council Doctoral Training Partnership studentship, Wellcome Trust, Medical Research Council (UK), French National Research Agency.