Secretion Of Virulence Proteins Research Articles

Antigen specificity shapes antibody functions in tuberculosis.

Tuberculosis (TB) is the number one infectious disease cause of death worldwide due to an incomplete understanding of immunity. Emerging data highlight antibody functions mediated by the Fc domain as immune correlates. However, the mechanisms by which antibody functions impact the causative agent Mycobacterium tuberculosis (Mtb) are unclear. Here, we examine how antigen specificity determined by the Fab domain shapes Fc effector functions against Mtb. Using the critical structural and secreted virulence proteins Mtb cell wall and ESAT-6 & CFP-10, we observe that antigen specificity alters subclass, antibody post-translational glycosylation, and Fc effector functions in TB patients. Moreover, Mtb cell wall IgG3 enhances disease through opsonophagocytosis of extracellular Mtb . In contrast, polyclonal and a human monoclonal IgG1 we generated targeting ESAT-6 & CFP-10 inhibit intracellular Mtb . These data show that antibodies have multiple roles in TB and antigen specificity is a critical determinant of the protective and pathogenic capacity.

The structural landscape and diversity of Pyricularia oryzae MAX effectors revisited.

Magnaporthe AVRs and ToxB-like (MAX) effectors constitute a family of secreted virulence proteins in the fungus Pyricularia oryzae (syn. Magnaporthe oryzae), which causes blast disease on numerous cereals and grasses. In spite of high sequence divergence, MAX effectors share a common fold characterized by a ß-sandwich core stabilized by a conserved disulfide bond. In this study, we investigated the structural landscape and diversity within the MAX effector repertoire of P. oryzae. Combining experimental protein structure determination and in silico structure modeling we validated the presence of the conserved MAX effector core domain in 77 out of 94 groups of orthologs (OG) identified in a previous population genomic study. Four novel MAX effector structures determined by NMR were in remarkably good agreement with AlphaFold2 (AF2) predictions. Based on the comparison of the AF2-generated 3D models we propose a classification of the MAX effectors superfamily in 20 structural groups that vary in the canonical MAX fold, disulfide bond patterns, and additional secondary structures in N- and C-terminal extensions. About one-third of the MAX family members remain singletons, without strong structural relationship to other MAX effectors. Analysis of the surface properties of the AF2 MAX models also highlights the high variability within the MAX family at the structural level, potentially reflecting the wide diversity of their virulence functions and host targets.

The WY Domain of an RxLr Effector Drives Interactions with a Host Target Phosphatase to Mimic Host Regulatory Proteins and Promote Phytophthora infestans Infection.

Plant pathogens manipulate the cellular environment of the host to facilitate infection and colonization that often lead to plant diseases. To accomplish this, many specialized pathogens secrete virulence proteins called effectors into the host cell, which subvert processes such as immune signaling, gene transcription, and host metabolism. Phytophthora infestans, the causative agent of potato late blight, employs an expanded repertoire of RxLR effectors with WY domains to manipulate the host through direct interaction with protein targets. However, our understanding of the molecular mechanisms underlying the interactions between WY effectors and their host targets remains limited. In this study, we performed a structural and biophysical characterization of the P. infestans WY effector Pi04314 in complex with the potato Protein Phosphatase 1-c (PP1c). We elucidate how Pi04314 uses a WY domain and a specialized C-terminal loop carrying a KVxF motif that interact with conserved surfaces on PP1c, known to be used by host regulatory proteins for guiding function. Through biophysical and in planta analyses, we demonstrate that Pi04314 WY or KVxF mutants lose their ability to bind PP1c. The loss of PP1c binding correlates with changes in PP1c nucleolar localization and a decrease in lesion size in plant infection assays. This study provides insights into the manipulation of plant hosts by pathogens, revealing how effectors exploit key regulatory interfaces in host proteins to modify their function and facilitate disease. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

In-silico Screening of Potential Phytochemicals against Extracellular Adherence (Eap) Protein of Staphylococcus aureus from Indian Medicinal Plants

Background: Staphylococcus aureus is a threat to human health, it colonizes one-third of the human population via skin or nose and deeper intrusions into tissues have catastrophic consequences. The bacterium secretes virulence proteins like CHIP and SCIN and extracellular adhesins like extracellular adherence (Eap) proteins. Eap and its functionally orphan homologs, EapH1 and EapH2; are a class of secreted proteins that inhibit neutrophil serine proteases such as neutrophil elastase (HNE) that is linked to tissue degradation in a variety of disease conditions including inflammatory disorders. Commercial drugs used against S.aureus such as Nafcillin, Pefloxacin, etc. have been known to have negative effects and are not recommended for children, the elderly, or pregnant women. Objective: The current research focuses on discovering phytochemicals found in Indian medicinal herbs that have been used as spices for ages and are already beneficial against a variety of illnesses and ailments to be used against Eap proteins. Method: Molecular docking;absorption, distribution, metabolism, excretion, toxicity (ADMET) analysis and Simulation were performed to see if these phytochemicals interact with the active site residues of Eap proteins and function as competitive inhibitors of NE and to know their drug like properties and gather information about the system dynamics. 19 phytochemicals were selected from receptor-ligand docking. The selected molecules were pharmacologically tested through Lipinski’s analysis; to know their ability for being formulated into drugs. ADMET analysis was carried out to define the biological characteristic of phytochemicals inside the living body. The phytochemicals with the best docking score and drug likeliness were analysed by Molecular Simulation to observe the fluctuation of participating and interacting amino acids with Eaph1 and Eaph2 respectively. Result: Among the nineteen phytochemicals that were chosen for docking only the best eleven interactions were chosen for ADMET analysis. The top hit phytochemicals for Eaph1 and Eaph2 were Curcumin and Eugenol respectively, which was well demonstrated by Molecular dynamic simulation. Conclusion: The present study has established the hypothesis that phytochemicals have a scope to replace commercial drugs against the Eap virulence system of Staphylococcus aureus

A receptor-like kinase SlFERL mediates immune responses of tomato to Botrytis cinerea by recognizing BcPG1 and fine-tuning MAPK signaling.

FERONIA (FER) is a receptor-like kinase showing versatile functions during plant growth, development, and responses to environmental stimuli. However, its functions during the interaction between fruit and necrotrophic fungal pathogens are still unclear. Combining reverse genetic approaches, physiological assays, co-immunoprecipitation, protein phosphorylation identification, and site-directed mutagenesis, we reported a tomato FER homolog SlFERL (Solanum lycopersicum FERONIA Like) involved in the immune responses to Botrytis cinerea invasion. The results indicated that SlFERL extracellular domain recognized and interacted with the secreted virulence protein BcPG1 from B. cinerea, further revealed that SlFERL triggered downstream signaling by phosphorylating SlMAP3K18 at Thr45, Ser49, Ser76, and Ser135. Moreover, we verified that SlMAP2K2 and SlMAP2K4 synergistically contributed to immune response of tomato to B. cinerea, in which SlFERL-SlMAP3K18 module substantially modulated protein level and/or kinase activity of SlMAP2K2/SlMAP2K4. These findings reveal a new pattern-triggered immune pathway, indicating that SlFERL participates in the immune responses to B. cinerea invasion via recognizing BcPG1 and fine-tuning MAPK signaling.

Plastic leachate exposure drives antibiotic resistance and virulence in marine bacterial communities

Plastic pollution is a serious global problem, with more than 12 million tonnes of plastic waste entering the oceans every year. Plastic debris can have considerable impacts on microbial community structure and functions in marine environments, and has been associated with an enrichment in pathogenic bacteria and antimicrobial resistance (AMR) genes. However, our understanding of these impacts is largely restricted to microbial assemblages on plastic surfaces. It is therefore unclear whether these effects are driven by the surface properties of plastics, providing an additional niche for certain microbes residing in biofilms, and/or chemicals leached from plastics, the effects of which could extend to surrounding planktonic bacteria. Here, we examine the effects of polyvinyl chloride (PVC) plastic leachate exposure on the relative abundance of genes associated with bacterial pathogenicity and AMR within a seawater microcosm community. We show that PVC leachate, in the absence of plastic surfaces, drives an enrichment in AMR and virulence genes. In particular, leachate exposure significantly enriches AMR genes that confer multidrug, aminoglycoside and peptide antibiotic resistance. Additionally, enrichment of genes involved in the extracellular secretion of virulence proteins was observed among pathogens of marine organisms. This study provides the first evidence that chemicals leached from plastic particles alone can enrich genes related to microbial pathogenesis within a bacterial community, expanding our knowledge of the environmental impacts of plastic pollution with potential consequences for human and ecosystem health.

Investigating the sequence properties that govern disorder and secretion of bacterial virulence proteins.

Many proteins contain disordered segments that play important roles in vivo. Autotransporter (AT) proteins are key virulence proteins from pathogenic Gram-negative bacteria. AT proteins have disordered properties that have been shown to be crucial for their efficient secretion to the bacterial cell surface. We have extensively studied pertactin, a virulence protein secreted by Bordetella pertussis, the bacterium that causes whooping cough. Like several other ATs, the mature pertactin virulence protein contains two segments with distinctly different folding properties. In isolation, the more stable C-terminal segment (PCt) is stably folded whereas the N-terminal segment (PNt) is disordered. Our previous studies indicate that folding of PCt is important for the efficient secretion of pertactin. Moreover, we recently showed that local clustering of hydrophobic residues leads to a significant collapse of PNt in vitro. Interestingly, we found that the extent to which PNt adopts a disordered, highly expanded conformational ensemble regulates the efficient secretion of pertactin through the bacterial outer membrane. Taken together, these observations suggest that PNt contains sequence properties important for secretion. Because disordered properties are important for AT secretion, understanding the connections between amino acid sequence features, disordered conformations, and AT secretion will improve our understanding of virulence protein secretion. The present research project aims to build upon our recent findings to develop a predictive understanding of the sequence factors that regulate disorder in pertactin and other proteins. We are using a combination of biophysical and biochemical approaches to characterize the sequence determinants of disorder and test their impact on AT secretion. We expect that insight into the regulation of disorder in pertactin will likely also apply to other members of the AT protein family, the largest family of secreted virulence proteins in Gram-negative bacterial pathogens.

Biodistribution of 89Zr-DFO-labeled avian pathogenic Escherichia coli outer membrane vesicles by PET imaging in chickens

Avian pathogenic Escherichia coli (APEC) is a serious systemic infectious disease in poultry infections, causing severe economic losses to the poultry industry. Previous studies have shown that secretion of virulence proteins was required for the pathogenicity of APEC through the secretion system. Outer membrane vesicles (OMVs) are a generalized secretion system of Gram-negative bacteria that play a key role in the long-distance delivery of virulence factors, but whether they are associated with the pathogenic mechanism of APEC has not been determined. In this study, OMVs were purified and characterized from AE17 (O2 serotype) by ultracentrifugation and density gradient centrifugation and their protein cargo was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, 89Zr was labeled after chelating AE17 OMVs by DFO and positron emission tomography PET imaging was used to track 89Zr-DFO-OMVs in chickens and to pathologically analyze the distribution sites. This study showed that AE17 OMVs were membrane vesicles ranging in size from 20 to 200 nm and proteomic analysis revealed the presence of virulence proteins, including adhesion proteins OmpA, OmpC, OmpF, OmpX, FimH, FimC and FigE, and serum resistance proteins OmpT and MliC and immune response regulator proteins (FliC). In addition, in vivo PET imaging to track the biodistribution of AE17 OMVs showed that AE17 OMVs were taken up by the lung region and the gastrointestinal and renal regions but were not detected in other areas. Pathological analysis of the tissue sites where AE17 OMVs were ingested showed inflammatory responses and damage. These findings suggested that AE17 OMVs not only contained a group of virulence proteins associated with AE17 infection but can also deliver these virulence proteins over long distances and caused tissue inflammatory damage. Our study revealed a previously unidentified causative microbial signal in the pathogenesis of APEC that could aid in the development of vaccines and antibiotics effective against APEC.

Secretome analysis of an environmental isolate Enterobacter sp. S-33 identifies proteins related to pathogenicity.

Enterobacter species are responsible for causing infections of the lower respiratory tract, urinary tract, meninges, etc. Proteins secreted by these species may act as determinants of host-pathogen interaction and play a role in virulence. Among the secreted proteins, the Type VI secretion system (T6SS) acts as a molecular nanomachine to deliver many effector proteins directly into prey cells in a contact-dependent manner. The secreted proteins may provide an idea for the interaction of bacteria to their environment and an understanding of the role of these proteins for their role in bacterial physiology and behaviour. Therefore, aim of this study was to characterize the secreted proteins in the culture supernatant by a T6SS bacterium Enterobacter sp. S-33 using nano-LC-MS/MS tool. Using a combined mass spectrometry and bioinformatics approach, we identified a total of 736 proteins in the secretome. Bioinformatics analysis predicting subcellular localization identified 110 of the secreted proteins possessed signal sequences. By gene ontology analysis, more than 80 proteins of the secretome were classified into biological or molecular functions. More than 20 percent of secretome proteins were virulence proteins including T6SS proteins, proteins involved in adherence and fimbriae formation, molecular chaperones, outer membrane proteins, serine proteases, antimicrobial, biofilm, exotoxins, etc. In summary, the results of the present study of the S-33 secretome provide a basis for understanding the possible pathogenic mechanisms and future investigation by detailed experimental approach will provide a confirmation of secreted virulence proteins in the exact role of virulence using the in vivo model.

Characterizing the sequence determinants of protein disorder and its impact on virulence protein secretion

Many proteins contain disordered segments that play important roles in vivo. Autotransporter (AT) proteins are key virulence proteins from pathogenic Gram-negative bacteria. AT proteins have disordered properties that have been shown to be crucial for their efficient secretion to the bacterial cell surface, but much remains unknown regarding the amino acid sequence properties that dictate protein disorder. In previous work using small angle X-ray scattering (SAXS), we showed that local clustering of hydrophobic residues leads to a significant collapse of a disordered segment of the AT protein pertactin (Bowman et al., (2020) PNAS), a virulence protein from Bordetella pertussis, the bacterium that causes whooping cough.

Unique virulence role of post-translocational chaperone PrsA in shaping Streptococcus pyogenes secretome

ABSTRACT Streptococcus pyogenes (group A Streptococcus, GAS) is a strict human pathogen causing a broad spectrum of diseases and a variety of autoimmune sequelae. The pathogenesis of GAS infection mostly relies on the production of an extensive network of cell wall-associated and secreted virulence proteins, such as adhesins, toxins, and exoenzymes. PrsA, the only extracellular parvulin-type peptidyl-prolyl isomerase expressed ubiquitously in Gram-positive bacteria, has been suggested to assist the folding and maturation of newly exported proteins to acquire their native conformation and activity. Two PrsA proteins, PrsA1 and PrsA2, have been identified in GAS, but the respective contribution of each PrsA in GAS pathogenesis remains largely unknown. By combining comparative proteomic and phenotypic analysis approaches, we demonstrate that both PrsA isoforms are required to maintain GAS proteome homeostasis and virulence-associated traits in a unique and overlapping manner. The inactivation of both PrsA in GAS caused remarkable impairment in biofilm formation, host adherence, infection-induced cytotoxicity, and in vivo virulence in a murine soft tissue infection model. The concordance of proteomic and phenotypic data clearly features the essential role of PrsA in GAS full virulence.

Yersinia pseudotuberculosis YopJ Limits Macrophage Response by Downregulating COX-2-Mediated Biosynthesis of PGE2 in a MAPK/ERK-Dependent Manner.

ABSTRACTProstaglandin E2 (PGE2) is an essential immunomodulatory lipid released by cells in response to infection with many bacteria, yet its function in macrophage-mediated bacterial clearance is poorly understood. Yersinia overall inhibits the inflammatory circuit, but its effect on PGE2 production is unknown. We hypothesized that one of the Yersinia effector proteins is responsible for the inhibition of PGE2 biosynthesis. We identified that yopB-deficient Y. enterocolitica and Y. pseudotuberculosis deficient in the secretion of virulence proteins via a type 3 secretion system (T3SS) failed to inhibit PGE2 biosynthesis in macrophages. Consistently, COX-2-mediated PGE2 biosynthesis is upregulated in cells treated with heat-killed or T3SS-deficient Y. pseudotuberculosis but diminished in the presence of a MAPK/ERK inhibitor. Mutants expressing catalytically inactive YopJ induce similar levels of PGE2 as heat-killed or ΔyopB Y. pseudotuberculosis, reversed by YopJ complementation. Shotgun proteomics discovered host pathways regulated in a YopJ-mediated manner, including pathways regulating PGE2 synthesis and oxidative phosphorylation. Consequently, this study identified that YopJ-mediated inhibition of MAPK signal transduction serves as a mechanism targeting PGE2, an alternative means of inflammasome inhibition by Yersinia. Finally, we showed that EP4 signaling supports macrophage function in clearing intracellular bacteria. In summary, our unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription, thereby enhancing the intracellular survival of yersiniae. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic molecule to improve the outcomes of specific bacterial infections. Since other pathogens encode YopJ homologs, this mechanism is expected to be present in other infections.IMPORTANCE PGE2 is a critical immunomodulatory lipid, but its role in bacterial infection and pathogen clearance is poorly understood. We previously demonstrated that PGE2 leads to macrophage polarization toward the M1 phenotype and stimulates inflammasome activation in infected macrophages. Finally, we also discovered that PGE2 improved the clearance of Y. enterocolitica. The fact that Y. enterocolitica hampers PGE2 secretion in a type 3 secretion system (T3SS)-dependent manner and because PGE2 appears to assist macrophage in the clearance of this bacterium indicates that targeting of the eicosanoid pathway by Yersinia might be an adaption used to counteract host defenses. Our study identified a mechanism used by Yersinia that obstructs PGE2 biosynthesis in human macrophages. We showed that Y. pseudotuberculosis interferes with PGE2 biosynthesis by using one of its T3SS effectors, YopJ. Specifically, YopJ targets the host COX-2 enzyme responsible for PGE2 biosynthesis, which happens in a MAPK/ER-dependent manner. Moreover, in a shotgun proteomics study, we also discovered other pathways that catalytically active YopJ targets in the infected macrophages. YopJ was revealed to play a role in limiting host LPS responses, including repression of EGR1 and JUN proteins, which control transcriptional activation of proinflammatory cytokine production such as interleukin-1β. Since YopJ has homologs in other bacterial species, there are likely other pathogens that target and inhibit PGE2 biosynthesis. In summary, our study’s unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic target.

"It Takes Two to Tango": Role of Neglected Macrophage Manipulators Coronin 1 and Protein Kinase G in Mycobacterial Pathogenesis.

Macrophages being the connecting link between innate and adaptive immune system plays a crucial role in microbial antigen presentation and orchestrates the subsequent clearance of microorganisms. Microbial invasion of macrophages trigger a plethora of signaling cascades, which interact among them to generate a dynamically altered hostile environment, that ultimately leads to disruption of microbial pathogenesis. Paradoxically, Mycobacterium sp. exploits macrophage proteins such as Coronin 1, Calcineurin, LRG47, SOCS1, CISH, Gbp5 etc. and secretes virulence proteins such as PknG, PtpA, SapM, Eis etc. to hijack these intra-macrophage, signaling cascades and thereby develop its own niche. Coronin 1, being a cortical protein is transiently recruited to all mycobacteria containing phagosomes, but only pathogenic mycobacteria can retain it on the phagosome, to hinder its maturation. Additionally, mycobacterial infection linked secretion of virulence factor Protein Kinase G through its phosphorylation, manipulates several macrophage signaling pathways and thus promotes pathogenesis at various stages, form early infection to latency to granuloma formation. Here we discuss the present status of mycobacteria engaged Coronin 1-dependent signaling cascades and secreted PknG related sequence of events promoting mycobacterial pathogenesis. Current knowledge about these two proteins in context of macrophage signaling manipulation encompassing diverse mechanisms like calcium-calcineurin signaling, reduced proinflamtory cytokine secretion, cytoskeletal changes, and adaptation in acidic environment, which ultimately converge toward mycobacterial survival inside the macrophages has been discussed.

GTP-Dependent FlhF Homodimer Supports Secretion of a Hemolysin in Bacillus cereus.

The multidomain (B-NG) protein FlhF, a flagellar biogenesis regulator in several bacteria, is the third paralog of the signal recognition particle (SRP)-GTPases Ffh and FtsY, which are known to drive protein-delivery to the plasma membrane. Previously, we showed that FlhF is required for Bacillus cereus pathogenicity in an insect model of infection, being essential for physiological peritrichous flagellation, for motility, and for the secretion of virulence proteins. Among these proteins, we found that the L2 component of hemolysin BL, one of the most powerful toxins B. cereus produces, was drastically reduced by the FlhF depletion. Herein, we demonstrate that B. cereus FlhF forms GTP-dependent homodimers in vivo since the replacement of residues critical for their GTP-dependent homodimerization alters this ability. The protein directly or indirectly controls flagellation by affecting flagellin-gene transcription and its overproduction leads to a hyperflagellated phenotype. On the other hand, FlhF does not affect the expression of the L2-encoding gene (hblC), but physically binds L2 when in its homodimeric form, recruiting the protein to the plasma membrane for secretion. We additionally show that FlhF overproduction increases L2 secretion and that the FlhF/L2 interaction requires the NG domain of FlhF. Our findings demonstrate the peculiar behavior of B. cereus FlhF, which is required for the correct flagellar pattern and acts as SRP-GTPase in the secretion of a bacterial toxin subunit.

Effects of Azole Fungicides on Secreted Metabolomes of Botrytis cinerea.

Botrytis cinerea, gray mold, is one of the most notorious phytopathgens, causing serious economic loss in the agricultural industry. The phytotoxic effects are mainly derived from secreted virulence proteins and terpenoid-type secondary metabolites. Azole fungicides are commonly used to manage the disease. However, their biochemical effects other than sterol biosynthesis were not documented, especially toxic secreted metabolites. In this study, six azole fungicides were treated with in vitro and in vivo conditions. Comprehensive profiles of primary and secondary metabolites in culture media were evaluated to assess the fungal metabolomes under pesticide-stressed conditions. The results indicated that extensive metabolic differentiation was induced by azole fungicides. Epoxiconazole clearly reduced the extracellular phytotoxin concentrations, while the level of indole-3-acetic acid was increased. In addition, significant differentiation of primary metabolism could be deduced from secreted metabolite profiles, including the tricarboxylic acid cycle and aromatic amino acid catabolism. Cellular lipid profiles, including fatty acids and sterol, have been altered drastically by azoles, which indicate extensive changes of cellular lipid metabolism. These system-wide metabolic alterations resulted in reduced plant damages, proven by the in vivo assay with tomato. Overall, azole fungicides induced significant changes of endo- and exometabolomes and could reduce the fungal infection. The experimental results will provide a more detailed understanding of physiological changes of phytopathogens under pesticide treatment and information for new pesticide development.

Influence of transcription regulator SAUSA300_1968 on the virulence protein secretion and immune evasion by Staphylococcus aureus

Staphylococcus aureus employs an arsenal of secreted virulence factors to evade host immune responses, such as inducing host cell death pathways or hiding within phagocytes. A previous study indicated that the transcription regulator SA1804 is a novel virulence repressor of S. aureus, and is regulated by SaeRS. However, the function of this regulator in S. aureus pathogenesis remains unclear. In this study, we created a SA1804 homologue, SAUSA300_1968, deletion mutant in the S. aureus USA300 strain LAC, and found that culture supernatant proteins of the mutant exhibit higher cytotoxicity and greater ability to induce the expression of inflammatory cytokines in mouse primary peritoneal macrophages as compared with that of its parent strain. Comparative proteome analysis of secreted protein expression profiles between the two strains was determined through tandem mass tags (TMT). Among the 18 well-known extracellular virulence-related factors that showed significantly different expressions, 17 proteins were up-regulated in the SAUSA300_1968 deletion mutant, including various pore-forming toxins and extracellular proteases. Accordingly, we also found that inactivation of SAUSA300_1968 enhanced S. aureus survival in mouse macrophages. Our data provide novel insights into the role of the SAUSA300_1968 regulator in host immune evasion and S. aureus pathogenesis.

Topological Analysis of the Type 3 Secretion System Translocon Pore Protein IpaC following Its Native Delivery to the Plasma Membrane during Infection.

Many Gram-negative bacterial pathogens require a type 3 secretion system (T3SS) to deliver effector proteins into eukaryotic cells. Contact of the tip complex of the T3SS with a target eukaryotic cell initiates secretion of the two bacterial proteins that assemble into the translocon pore in the plasma membrane. The translocon pore functions to regulate effector protein secretion and is the conduit for effector protein translocation across the plasma membrane. To generate insights into how the translocon pore regulates effector protein secretion, we defined the topology of the Shigella translocon pore protein IpaC in the plasma membrane following its native delivery by the T3SS. Using single cysteine substitution mutagenesis and site-directed labeling with a membrane-impermeant chemical probe, we mapped residues accessible from the extracellular surface of the cell. Our data support a model in which the N terminus of IpaC is extracellular and the C terminus of IpaC is intracellular. These findings resolve previously conflicting data on IpaC topology that were based on nonnative delivery of IpaC to membranes. Salmonella enterica serovar Typhimurium also requires the T3SS for effector protein delivery into eukaryotic cells. Although the sequence of IpaC is closely related to the Salmonella translocon pore protein SipC, the two proteins have unique functional attributes during infection. We showed a similar overall topology for SipC and IpaC and identified subtle topological differences between their transmembrane α-helixes and C-terminal regions. Together, our data suggest that topological differences among distinct translocon pore proteins may dictate organism-specific functional differences of the T3SSs during infection.IMPORTANCE The type 3 secretion system (T3SS) is a nanomachine required for virulence of many bacterial pathogens that infect humans. The system delivers bacterial virulence proteins into the cytosol of human cells, where the virulence proteins promote bacterial infection. The T3SS forms a translocon pore in the membranes of target cells. This pore is the portal through which bacterial virulence proteins are delivered by the T3SS into the eukaryotic cytosol. The pore also regulates secretion of these virulence proteins. Our work defines the topology of translocon pore proteins in their native context during infection, resolves previously conflicting reports about the topology of the Shigella translocon pore protein IpaC, and provides new insights into how interactions of the pore with the T3SS likely produce signals that activate secretion of virulence proteins.

How many protein molecules are secreted by single Helicobacter pylori cells: Quantification of serine protease HtrA.

Infection with Helicobacter pylori represents a major risk for developing peptic ulcer disease, gastric adenocarcinoma, and various other gastric and nongastric sicknesses. A series of H.pylori virulence factors can be secreted into the cell culture supernatant, and the secretome contains more than 100 different proteins. However, the quantities of proteins secreted by the bacteria over time are unknown. One of these factors is the serine protease high-temperature requirement A (HtrA), encoded by an essential bifunctional gene with crucial intracellular and extracellular activities. We have demonstrated recently that secreted HtrA can cleave off the ectodomains of the tight junction proteins occludin and claudin-8, as well as of the tumour suppressor and adherens junction protein E-cadherin on polarised gastric epithelial cells. The exact mechanism of secretion and the quantity of secreted HtrA, however, have not been studied in detail. Here, we applied protein purification and quantitative Western blotting to determine the number of HtrA molecules secreted by H.pylori cells in liquid culture during a time course. Over a period of 8hr, actively dividing bacteria secreted HtrA at a similar rate, on average about 9,600 HtrA molecules per cell. We determined minor variation over time corresponding to 9,931±1,768 at an OD600 of 0.4 after 2hr, 9,403±2,356 2hr later, and 9,644±2,067 molecules per cell after 8hr of culturing, when the culture had reached an OD600 of 0.8. This is the first report on the quantification of a secreted virulence protein from the important gastric pathogen H.pylori. Because HtrA has been considered as a promising new target for antibacterial therapy, knowledge about secreted protein quantities is crucial for optimising corresponding treatment regimes.

A Multicolor Split-Fluorescent Protein Approach to Visualize Listeria Protein Secretion in Infection

Listeria monocytogenes is an intracellular food-borne pathogen that has evolved to enter mammalian host cells, survive within them, spread from cell to cell, and disseminate throughout the body. A series of secreted virulence proteins from Listeria are responsible for manipulation of host-cell defense mechanisms and adaptation to the intracellular lifestyle. Identifying when and where these virulence proteins are located in live cells over the course of Listeria infection can provide valuable information on the roles these proteins play in defining the host-pathogen interface. These dynamics and protein levels may vary from cell to cell, as bacterial infection is a heterogeneous process both temporally and spatially. No assay to visualize virulence proteins over time in infection with Listeria or other Gram-positive bacteria has been developed. Therefore, we adapted a live, long-term tagging system to visualize a model Listeria protein by fluorescence microscopy on a single-cell level in infection. This system leverages split-fluorescent proteins, in which the last strand of a fluorescent protein (a 16-amino-acid peptide) is genetically fused to the virulence protein of interest. The remainder of the fluorescent protein is produced in the mammalian host cell. Both individual components are nonfluorescent and will bind together and reconstitute fluorescence upon virulence-protein secretion into the host cell. We demonstrate accumulation and distribution within the host cell of the model virulence protein InlC in infection over time. A modular expression platform for InlC visualization was developed. We visualized InlC by tagging it with red and green split-fluorescent proteins and compared usage of a strong constitutive promoter versus the endogenous promoter for InlC production. This split-fluorescent protein approach is versatile and may be used to investigate other Listeria virulence proteins for unique mechanistic insights in infection progression.

Enteropathogenic Escherichia coli EspH-Mediated Rho GTPase Inhibition Results in Desmosomal Perturbations.

Background & AimsThe diarrheagenic pathogen, enteropathogenic Escherichia coli (EPEC), uses a type III secretion system to deliver effector molecules into intestinal epithelial cells (IECs). While exploring the basis for the lateral membrane separation of EPEC-infected IECs, we observed infection-induced loss of the desmosomal cadherin desmoglein-2 (DSG2). We sought to identify the molecule(s) involved in, and delineate the mechanisms and consequences of, EPEC-induced DSG2 loss.MethodsDSG2 abundance and localization was monitored via immunoblotting and immunofluorescence, respectively. Junctional perturbations were visualized by electron microscopy, and cell–cell adhesion was assessed using dispase assays. EspH alanine-scan mutants as well as pharmacologic agents were used to evaluate impacts on desmosomal alterations. EPEC-mediated DSG2 loss, and its impact on bacterial colonization in vivo, was assessed using a murine model.ResultsThe secreted virulence protein EspH mediates EPEC-induced DSG2 degradation, and contributes to desmosomal perturbation, loss of cell junction integrity, and barrier disruption in infected IECs. EspH sequesters Rho guanine nucleotide exchange factors and inhibits Rho guanosine triphosphatase signaling; EspH mutants impaired for Rho guanine nucleotide exchange factor interaction failed to inhibit RhoA or deplete DSG2. Cytotoxic necrotizing factor 1, which locks Rho guanosine triphosphatase in the active state, jasplakinolide, a molecule that promotes actin polymerization, and the lysosomal inhibitor bafilomycin A, respectively, rescued infected cells from EPEC-induced DSG2 loss. Wild-type EPEC, but not an espH-deficient strain, colonizes mouse intestines robustly, widens paracellular junctions, and induces DSG2 re-localization in vivo.ConclusionsOur studies define the mechanism and consequences of EPEC-induced desmosomal alterations in IECs. These perturbations contribute to the colonization and virulence of EPEC, and likely related pathogens.