A potential virulence factor: Brucella flagellin FliK does not affect the main biological properties but inhibits the inflammatory response in RAW264.7 cells.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

Alterations in cell arrangements of group B streptococcus due to virulence factor expression can bias estimates of bacterial populations based on colony count measures.

Group B streptococcus (GBS) is a chain-forming commensal bacterium and opportunistic pathogen that resides in the gastrointestinal and genitourinary tract of healthy adults. GBS can cause various infections and related complications in pregnant and nonpregnant women, adults, and newborns. Investigations of the mechanisms by which GBS causes disease pathogenesis often utilize colony count assays to estimate bacterial population size in experimental models. In other streptococci, such as group A streptococcus and pneumococcus, variation in the chain length of the bacteria that can occur naturally or due to mutation can affect facets of pathogenesis, such as adherence to or colonization of a host. No studies have reported a relationship between GBS chain length and pathogenicity. Here, we used GBS strain 874391 and several derivative strains displaying longer chain-forming phenotypes (874391pgapC, 874391ΔcovR, 874391Δstp1) to assess the impact of chain length on bacterial population estimates based on the colony-forming unit (c.f.u.) assay. Disruption of GBS chains via bead beating or sonication in conjunction with fluorescence microscopy was used to compare chaining phenotypes pre- and post-disruption to detect long- and short-chain forms, respectively. We used a murine model of GBS colonization of the female reproductive tract to assess whether chaining may affect bacterial colonization dynamics in the host during chronic infection in vivo. Overall, we found that GBS exhibiting long-chain form can significantly affect population size estimates based on the colony count assay. Additionally, we found that the length of chaining of GBS can affect virulence in the reproductive tract colonization model. Collectively, these findings have implications for studies of GBS that utilize colony count assays to measure GBS populations and establish that chain length can affect infection dynamics and disease pathogenesis for this important opportunistic pathogen.

GRASP negatively regulates the secretion of the virulence factor gp63 in Leishmania.

Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.

Thymoquinone is a natural antibiofilm and pathogenicity attenuating agent in Pseudomonas aeruginosa.

Pseudomonas aeruginosa belongs to the critical pathogens that represent a global public health problem due to their high rate of resistance as listed by WHO. P. aeruginosa can result in many nosocomial infections especially in individuals with compromised immune systems. Attenuating virulence factors by interference with quorum sensing (QS) systems is a promising approach to treat P. aeruginosa-resistant infections. Thymoquinone is a natural compound isolated from Nigella sativa (black seed) essential oil. In this study, the minimum inhibitory concentration of thymoquinone was detected followed by investigating the antibiofilm and antivirulence activities of the subinhibitory concentration of thymoquinone against P. aeruginosa PAO1. The effect of thymoquinone on the expression of QS genes was assessed by quantitative real-time PCR, and the protective effect of thymoquinone against the pathogenesis of PAO1 in mice was detected by the mouse survival test. Thymoquinone significantly inhibited biofilm, pyocyanin, protease activity, and swarming motility. At the molecular level, thymoquinone markedly downregulated QS genes lasI, lasR, rhlI, and rhlR. Moreover, thymoquinone could protect mice from the pathologic effects of P. aeruginosa increasing mouse survival from 20% to 100%. In conclusion, thymoquinone is a promising natural agent that can be used as an adjunct therapeutic agent with antibiotics to attenuate the pathogenicity of P. aeruginosa.

Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood-brain barrier damage in ducks during Riemerella anatipestifer infection.

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

Quenching of quorum sensing in multi-drug resistant Pseudomonas aeruginosa: insights on halo-bacterial metabolites and gamma irradiation as channels inhibitors.

BACKGROUND: Anti-virulence therapy is a promising strategy to treat multi-drug resistant (MDR) pathogens. Pseudomonas aeruginosa is a potent opportunistic pathogen because of an array of virulence factors that are regulated by quorum sensing systems. METHODS: The virulence features of four multi-drug resistant P. aeruginosa strains were investigated upon exposure to the sub-lethal dose of gamma rays (1 kGy), and sub-inhibitory concentrations of bioactive metabolites recovered from local halophilic strains in comparison to control. Then, the gene expression of AHL-mediated quorum sensing systems (las/rhl) was quantitatively determined in treated and untreated groups by real-time PCR. RESULTS: The bioactive metabolites recovered from halophilic strains previously isolated from saline ecosystems were identified as Halomonas cupida (Halo-Rt1), H. elongate (Halo-Rt2), Vigibacillus natechei (Halo-Rt3), Sediminibacillus terrae (Halo-Rt4) and H. almeriensis (Halo-Rt5). Results revealed that both gamma irradiation and bioactive metabolites significantly reduced the virulence factors of the tested MDR strains. The bioactive metabolites showed a maximum efficiency for inhibiting biofilm formation and rhamnolipids production whereas the gamma irradiation succeeded in decreasing other virulence factors to lower levels in comparison to control. Quantitative-PCR results showed that AHL-mediated quorum sensing systems (las/rhl) in P. aeruginosa strains were downregulated either by halo-bacterial metabolites or gamma irradiation in all treatments except the upregulation of both lasI internal gene and rhlR intact gene in P. aeruginosa NCR-RT3 and both rhlI internal gene and rhlR intact gene in P. aeruginosa U3 by nearly two folds or more upon exposure to gamma irradiation. The most potent result was observed in the expression of lasI internal gene that was downregulated by more than ninety folds in P. aeruginosa NCR-RT2 after treatment with metabolites of S. terrae (Halo-Rt4). Analyzing metabolites recovered from H. cupida (Halo-Rt1) and H. elongate (Halo-Rt2) using LC-ESI-MS/MS revealed many chemical compounds that have quorum quenching properties including glabrol, 5,8-dimethoxyquinoline-2-carbaldehyde, linoleoyl ethanolamide, agelasine, penigequinolones derivatives, berberine, tetracosanoic acid, and liquidambaric lactone in the former halophile and phloretin, lycoctonine, fucoxanthin, and crassicauline A in the latter one. CONCLUSION: QS inhibitors can significantly reduce the pathogenicity of MDR P. aeruginosa strains; and thus can be an effective and successful strategy for treating antibiotic resistant traits.

Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins.

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.

Comprehensive Review on the Virulence Factors and Therapeutic Strategies with the Aid of Artificial Intelligence against Mucormycosis.

Mucormycosis, a rare but deadly fungal infection, was an epidemic during the COVID-19 pandemic. The rise in cases (COVID-19-associated mucormycosis, CAM) is attributed to excessive steroid and antibiotic use, poor hospital hygiene, and crowded settings. Major contributing factors include diabetes and weakened immune systems. The main manifesting forms of CAM─cutaneous, pulmonary, and the deadliest, rhinocerebral─and disseminated infections elevated mortality rates to 85%. Recent focus lies on small-molecule inhibitors due to their advantages over standard treatments like surgery and liposomal amphotericin B (which carry several long-term adverse effects), offering potential central nervous system penetration, diverse targets, and simpler dosing owing to their small size, rendering the ability to traverse the blood-brain barrier via passive diffusion facilitated by the phospholipid membrane. Adaptation and versatility in mucormycosis are facilitated by a multitude of virulence factors, enabling the pathogen to dynamically respond to various environmental stressors. A comprehensive understanding of these virulence mechanisms is imperative for devising effective therapeutic interventions against this highly opportunistic pathogen that thrives in immunocompromised individuals through its angio-invasive nature. Hence, this Review delineates the principal virulence factors of mucormycosis, the mechanisms it employs to persist in challenging host environments, and the current progress in developing small-molecule inhibitors against them.

Interaction between Yersinia pestis Ail Outer Membrane Protein and the C-Terminal Domain of Human Vitronectin.

Yersinia pestis, the causative agent of plague, is capable of evading the human immune system response by recruiting the plasma circulating vitronectin proteins, which act as a shield and avoid its lysis. Vitronectin recruitment is mediated by its interaction with the bacterial transmembrane protein Ail, protruding from the Y. pestis outer membrane. By using all-atom long-scale molecular dynamic simulations of Ail embedded in a realistic model of the bacterial membrane, we have shown that vitronectin forms a stable complex, mediated by interactions between the disordered moieties of the two proteins. The main amino acids driving the complexation have also been evidenced, thus favoring the possible rational design of specific peptides which, by inhibiting vitronectin recruitment, could act as original antibacterial agents.

Unveiling the ocular battlefield: Insights into Pseudomonas aeruginosa virulence factors and their implications for multidrug resistance.

The research investigates the virulence factors of Pseudomonas aeruginosa (P. aeruginosa), a pathogen known for its ability to cause human infections by releasing various exoenzymes and virulence factors. Particularly relevant in ocular infections, where tissue degeneration can occur, even after bacterial growth has ceased due to the potential role of secreted proteins/enzymes. Clinical isolates of P. aeruginosa, both ocular (146) and non-ocular (54), were examined to determine the frequency and mechanism of virulence factors. Phenotypic characterization revealed the production of alginate, biofilm, phospholipase C, and alkaline protease, while genotypic testing using internal uniplex PCR identified the presence of Exo U, S, T, Y, and LasB genes. Results showed a significant prevalence of Exo U and Y genes in ocular isolates, a finding unique to Indian studies. Additionally, the study noted that ocular isolates often contained all four secretomes, suggesting a potential link between these factors and ocular infections. These findings contribute to understanding the pathogenesis of P. aeruginosa infections, particularly in ocular contexts, and highlights the importance of comprehensive virulence factor analysis in clinical settings.

Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

Unveiling the pathogenic mechanisms of Clostridium perfringens toxins and virulence factors.

Clostridium perfringens causes multiple diseases in humans and animals. Its pathogenic effect is supported by a broad and heterogeneous arsenal of toxins and other virulence factors associated with a specific host tropism. Molecular approaches have indicated that most C. perfringens toxins produce membrane pores, leading to osmotic cell disruption and apoptosis. However, identifying mechanisms involved in cell tropism and selective toxicity effects should be studied more. The differential presence and polymorphisms of toxin-encoding genes and genes encoding other virulence factors suggest that molecular mechanisms might exist associated with host preference, receptor binding, and impact on the host; however, this information has not been reviewed in detail. Therefore, this review aims to clarify the current state of knowledge on the structural features and mechanisms of action of the major toxins and virulence factors of C. perfringens and discuss the impact of genetic diversity of toxinotypes in tropism for several hosts.

Functional Divergence of the Paralog Salmonella Effector Proteins SopD and SopD2 and Their Contributions to Infection.

Salmonella enterica is a leading cause of bacterial food-borne illness in humans and is responsible for millions of cases annually. A critical strategy for the survival of this pathogen is the translocation of bacterial virulence factors termed effectors into host cells, which primarily function via protein-protein interactions with host proteins. The Salmonella genome encodes several paralogous effectors believed to have arisen from duplication events throughout the course of evolution. These paralogs can share structural similarities and enzymatic activities but have also demonstrated divergence in host cell targets or interaction partners and contributions to the intracellular lifecycle of Salmonella. The paralog effectors SopD and SopD2 share 63% amino acid sequence similarity and extensive structural homology yet have demonstrated divergence in secretion kinetics, intracellular localization, host targets, and roles in infection. SopD and SopD2 target host Rab GTPases, which represent critical regulators of intracellular trafficking that mediate diverse cellular functions. While SopD and SopD2 both manipulate Rab function, these paralogs display differences in Rab specificity, and the effectors have also evolved multiple mechanisms of action for GTPase manipulation. Here, we highlight this intriguing pair of paralog effectors in the context of host-pathogen interactions and discuss how this research has presented valuable insights into effector evolution.

The Staphylococcus aureus regulatory program in a human skin-like environment.

Staphylococcus aureus is a Gram-positive pathogen responsible for the majority of skin and soft tissue infections (SSTIs). S. aureus colonizes the anterior nares of approximately 20%-30% of the population and transiently colonizes the skin, thereby increasing the risk of developing SSTIs and more serious infections. Current laboratory models that mimic the skin surface environment are expensive, require substantial infrastructure, and limit the scope of bacterial physiology studies under human skin conditions. To overcome these limitations, we developed a cost-effective, open-source, chemically defined media recipe termed skin-like medium (SLM) that incorporates key aspects of the human skin surface environment and supports growth of several staphylococcal species. We utilized SLM to investigate the transcriptional response of methicillin-resistant Staphylococcus aureus (MRSA) following growth in SLM compared to a commonly used laboratory media. Through RNA-seq analysis, we observed the upregulation of several virulence factors, including genes encoding functions involved in adhesion, proteolysis, and cytotoxicity. To further explore these findings, we conducted quantitative reverse transcription-PCR (qRT-PCR) experiments to determine the influence of media composition, pH, and temperature on the transcriptional response of key factors involved in adhesion and virulence. We also demonstrated that MRSA primed in SLM adhered better to human corneocytes and demonstrated adhesin-specific phenotypes that previously required genetic manipulation. This improved adherence to corneocytes was dependent on both acidic pH and growth in SLM. These results support the potential utility of SLM as an in vitro model for assessing staphylococcal physiology and metabolism on human skin. IMPORTANCE: Staphylococcus aureus is the major cause of skin diseases, and its increased prevalence in skin colonization and infections present a need to understand its physiology in this environment. The work presented here outlines S. aureus upregulation of colonization and virulence factors using a newly developed medium that strives to replicate the human skin surface environment and demonstrates roles for adhesins clumping factor A (ClfA), serine-rich repeat glycoprotein adhesin (SraP), and the fibronectin binding proteins (Fnbps) in human corneocyte adherence.

Analyzing the safety of the parasiticide fungus Mucor circinelloides: first insights on its virulence profile and interactions with the avian gut microbial community.

Parasiticide fungi are considered an accurate, sustainable, and safe solution for the biocontrol of animal gastrointestinal (GI) parasites. This research provides an initial characterization of the virulence of the native parasiticide fungus Mucor circinelloides (FMV-FR1) and an assessment of its impact on birds' gut microbes. The genome of this fungus was sequenced to identify the genes coding for virulence factors. Also, this fungus was checked for the phenotypic expression of proteinase, lecithinase, DNase, gelatinase, hemolysin, and biofilm production. Finally, an in vivo trial was developed based on feeding M. circinelloides spores to laying hens and peacocks three times a week. Bird feces were collected for 3 months, with total genomic DNA being extracted and subjected to long-read 16S and 25S-28S sequencing. Genes coding for an iron permease (FTR1), iron receptors (FOB1 and FOB2), ADP-ribosylation factors (ARFs) (ARF2 and ARF6), and a GTPase (CDC42) were identified in this M. circinelloides genome. Also, this fungus was positive only for lecithinase activity. The field trial revealed a fecal microbiome dominated by Firmicutes and Proteobacteria in laying hens, and Firmicutes and Bacteroidetes in peacocks, whereas the fecal mycobiome of both bird species was mainly composed of Ascomycetes and Basidiomycetes fungi. Bacterial and fungal alpha-diversities did not differ between sampling time points after M. circinelloides administrations (P = 0.62 and P = 0.15, respectively). Although findings from this research suggest the lack of virulence of this M. circinelloides parasiticide isolate, more complementary in vitro and in vivo research is needed to conclude about the safety of its administration to birds, aiming at controlling their GI parasites.IMPORTANCEA previous study revealed that the native Mucor circinelloides isolate (FMV-FR1) can develop parasiticide activity toward coccidia oocysts, one of the most pathogenic GI parasites in birds. However, ensuring its safety for birds is of utmost importance, namely by studying its virulence profile and potential effect on commensal gut microbes. This initial study revealed that although this M. circinelloides isolate had genes coding for four types of virulence factors-iron permease, iron receptors, ADP-ribosylation factors, and GTPase-and only expressed phenotypically the enzyme lecithinase, the administration of its spores to laying hens and peacocks did not interfere with the abundances and diversities of their gut commensal bacteria and fungi. Although overall results suggest the lack of virulence of this M. circinelloides isolate, more complementary research is needed to conclude about the safety of its administration to birds in the scope of parasite biocontrol programs.

Development of a sensor for disulfide bond formation in diverse bacteria.

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In Escherichia coli, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins, while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR, which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/VKOR, represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using E. coli cells expressing a periplasmic ß-Galactosidase sensor (ß-Galdbs), which is only active when disulfide bond formation is inhibited. Here, we report the construction of plasmids that allows fine-tuning of the expression of the ß-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when expressed in Pseudomonas aeruginosa UCBPP-PA14, which harbors two DsbB homologs, ß-Galdbs behaves similarly as in E. coli, and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these ß-Galdbs reporter plasmids provide a basis to identify novel inhibitors of DsbA and DsbB/VKOR in multidrug-resistant gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria. IMPORTANCE: Disulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria because inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.

The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of this pathogen is type III secretion system 2 (T3SS2), which is encoded on the 80-kb V. parahaemolyticus pathogenicity island (Vp-PAI), the gene expression of which is governed by the OmpR-family transcriptional regulator VtrB. Here, we found a positive autoregulatory feature of vtrB transcription, which is often observed with transcriptional regulators of bacteria, but the regulation was not canonically dependent on its own promoter. Instead, this autoactivation was induced by heterogeneous transcripts derived from the VtrB-regulated operon upstream of vtrB. VtrB-activated transcription overcame the intrinsic terminator downstream of the operon, resulting in transcription read-through with read-in transcription of the vtrB gene and thus completing the autoregulatory loop for vtrB gene expression. The dampening of read-through transcription with an exogenous strong terminator reduced vtrB gene expression. Furthermore, a V. parahaemolyticus mutant with defects in the vtrB autoregulatory loop also showed compromises in T3SS2 expression and T3SS2-dependent cytotoxicity in vitro and enterotoxicity in vivo, indicating that this autoregulatory loop is essential for sustained vtrB activation and the consequent robust expression of T3SS2 genes for pathogenicity. Taken together, these findings demonstrate that the regulatory loop for vtrB gene expression based on read-through transcription from the upstream operon is a crucial pathway in T3SS2 gene regulatory network to ensure T3SS2-mediated virulence of V. parahaemolyticus.

Uncovering a hidden functional role of the XRE-cupin protein PsdR as a novel quorum-sensing regulator in Pseudomonas aeruginosa.

XRE-cupin family proteins containing an DNA-binding domain and a cupin signal-sensing domain are widely distributed in bacteria. In Pseudomonas aeruginosa, XRE-cupin transcription factors have long been recognized as regulators exclusively controlling cellular metabolism pathways. However, their potential functional roles beyond metabolism regulation remain unknown. PsdR, a typical XRE-cupin transcriptional regulator, was previously characterized as a local repressor involved solely in dipeptide metabolism. Here, by measuring quorum-sensing (QS) activities and QS-controlled metabolites, we uncover that PsdR is a new QS regulator in P. aeruginosa. Our RNA-seq analysis showed that rather than a local regulator, PsdR controls a large regulon, including genes associated with both the QS circuit and non-QS pathways. To unveil the underlying mechanism of PsdR in modulating QS, we developed a comparative transcriptome approach named "transcriptome profile similarity analysis" (TPSA). Using this TPSA method, we revealed that PsdR expression causes a QS-null-like transcriptome profile, resulting in QS-inactive phenotypes. Based on the results of TPSA, we further demonstrate that PsdR directly binds to the promoter for the gene encoding the QS master transcription factor LasR, thereby negatively regulating its expression and influencing QS activation. Moreover, our results showed that PsdR functions as a negative virulence regulator, as inactivation of PsdR enhanced bacterial cytotoxicity on host cells. In conclusion, we report on a new QS regulation role for PsdR, providing insights into its role in manipulating QS-controlled virulence. Most importantly, our findings open the door for a further discovery of untapped functions for other XRE-Cupin family proteins.

Physiological adventures in Candida albicans: farnesol and ubiquinones.

SUMMARYFarnesol was first identified as a quorum-sensing molecule, which blocked the yeast to hyphal transition in Candida albicans, 22 years ago. However, its interactions with Candida biology are surprisingly complex. Exogenous (secreted or supplied) farnesol can also act as a virulence factor during pathogenesis and as a fungicidal agent triggering apoptosis in other competing fungi. Farnesol synthesis is turned off both during anaerobic growth and in opaque cells. Distinctly different cellular responses are observed as exogenous farnesol levels are increased from 0.1 to 100 µM. Reported changes include altered morphology, stress response, pathogenicity, antibiotic sensitivity/resistance, and even cell lysis. Throughout, there has been a dearth of mechanisms associated with these observations, in part due to the absence of accurate measurement of intracellular farnesol levels (Fi). This obstacle has recently been overcome, and the above phenomena can now be viewed in terms of changing Fi levels and the percentage of farnesol secreted. Critically, two aspects of isoprenoid metabolism present in higher organisms are absent in C. albicans and likely in other yeasts. These are pathways for farnesol salvage (converting farnesol to farnesyl pyrophosphate) and farnesylcysteine cleavage, a necessary step in the turnover of farnesylated proteins. Together, these developments suggest a unifying model, whereby high, threshold levels of Fi regulate which target proteins are farnesylated or the extent to which they are farnesylated. Thus, we suggest that the diversity of cellular responses to farnesol reflects the diversity of the proteins that are or are not farnesylated.

Laser desorption rapid evaporative ionization mass spectrometry (LD-REIMS) demonstrates a direct impact of hypochlorous acid stress on PQS-mediated quorum sensing in Pseudomonas aeruginosa.

To establish infections in human hosts, Pseudomonas aeruginosa must overcome innate immune-generated oxidative stress, such as the hypochlorous acid (HOCl) produced by neutrophils. We set out to find specific biomarkers of oxidative stress through the development of a protocol for the metabolic profiling of P. aeruginosa cultures grown in the presence of different oxidants using a novel ionization technique for mass spectrometry, laser desorption rapid evaporative ionization mass spectrometry (LD-REIMS). We demonstrated the ability of LD-REIMS to classify samples as untreated or treated with a specific oxidant with 100% accuracy and identified a panel of 54 metabolites with significantly altered concentrations after exposure to one or more of the oxidants. Key metabolic changes were conserved in P. aeruginosa clinical strains isolated from patients with cystic fibrosis lung infections. These data demonstrated that HOCl stress impacted the Pseudomonas quinolone signal (PQS) quorum sensing system. Ten 2-alkyl-4-quinolones (AHQs) associated with the PQS system were significantly lower in concentration in HOCl-stressed P. aeruginosa cultures, including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), the most active signal molecule of the PQS system. The PQS system regulates the production of virulence factors, including pyocyanin and elastase, and their levels were markedly affected by HOCl stress. No pyocyanin was detectable and elastase concentrations were reduced by more than 75% in cultures grown with sub-lethal concentrations of HOCl, suggesting that this neutrophil-derived oxidant may disrupt the ability of P. aeruginosa to establish infections through interference with production of PQS-associated virulence factors. IMPORTANCE: This work demonstrates that a high-throughput ambient ionization mass spectrometry method can be used successfully to study a bacterial stress response. Its application to the opportunistic pathogen Pseudomonas aeruginosa led to the identification of specific oxidative stress biomarkers, and demonstrated that hypochlorous acid, an oxidant specifically produced by human neutrophils during infection, affects quorum sensing and reduces production of the virulence factors pyocyanin and elastase. No pyocyanin was detectable and elastase levels were reduced by more than 75% in bacteria grown in the presence of hypochlorous acid. This approach has the potential to be widely applicable to the characterization of the stress responses of bacteria.