Unravelling the role of anaerobic metabolism (pta-ackA) and virulence (misL and ssa) genes in Salmonella Heidelberg shedding using chicken infection model.

The mechanism of colonisation of the chicken intestine by Salmonella remains poorly understood, while the severity of infections vary enormously depending on the serovar and the age of the bird. Several metabolism and virulence genes have been identified in Salmonella Heidelberg; however, information on their roles in infection, particularly in the chicken infection model, remains scarce. In the present publication, we investigated three Salmonella Heidelberg mutants containing deletions in misL, ssa, and pta-ackA genes by using signature-tagged mutagenesis. We found that mutations in these genes of S. Heidelberg result in an increase in fitness in the chicken model. The exception was perhaps the pta-ackA mutant where colonisation was slightly reduced (2, 7, 14, and 21 days post-infection) although some birds were still excreting at the end of the experiment. Our results suggest that for intestinal colonisation of the chicken caecum, substrate-level phosphorylation is likely to be more important than the MisL outer membrane protein or even the secretion system apparatus. These findings validate previous work that demonstrated the contribution of ackA and pta mutants to virulence in chickens, suggesting that the anaerobic metabolism genes such as pta-ackA could be a promising mitigation strategy to reduce S. Heidelberg virulence.

Complete Genome Sequence of Pantoea stewartii RON18713 from Brazil Nut Tree Phyllosphere Reveals Genes Involved in Plant Growth Promotion.

The Amazonian rainforest is a hyper-diverse ecosystem in the number of species and the myriad of intertaxon relationships that are mostly understudied. In order to characterize a dominant and economically important Amazonian species, the Brazil nut tree (Bertholletia excelsa Bonpl.), at the genome level, wegenerated high-coverage long-read sequencing data from the leaves of a single individual. The genome assembly revealed an unexpected discovery: two circular contigs that could be assigned to the chromosome and a plasmid of a Pantoea stewartii strain. Comparative genomics revealed that this strain belongs to the indologenes subspecies and displays high synteny with other strains isolated from diseased leaves of the neotropical palm Bactris gasipaes Kunth. Investigation of pathogenicity-related genes revealed the absence of the entire type III secretion system gene cluster in the plasmid, which was otherwise highly similar to a plasmid from an isolate known to cause disease in Dracaena sanderiana Mast. In contrast, several genes associated with plant-growth promoting traits were detected, including genes involved in indole-3-acetic acid (IAA) production, phosphate solubilization, and biosynthesis of siderophores. In summary, we report the genome of an uncultivated P. stewartii subsp. indologenes strain associated with the Brazil nut tree and potentially a plant growth-promoting bacteria.

Vibrio type III secretion system 2 is not restricted to the Vibrionaceae and encodes differentially distributed repertoires of effector proteins.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. A distinctive feature of the O3:K6 pandemic clone, and its derivatives, is the presence of a second, phylogenetically distinct, type III secretion system (T3SS2) encoded within the genomic island VPaI-7. The T3SS2 allows the delivery of effector proteins directly into the cytosol of infected eukaryotic cells to subvert key host-cell processes, critical for V. parahaemolyticus to colonize and cause disease. Furthermore, the T3SS2 also increases the environmental fitness of V. parahaemolyticus in its interaction with bacterivorous protists; hence, it has been proposed that it contributed to the global oceanic spread of the pandemic clone. Several reports have identified T3SS2-related genes in Vibrio and non-Vibrio species, suggesting that the T3SS2 gene cluster is not restricted to the Vibrionaceae and can mobilize through horizontal gene transfer events. In this work, we performed a large-scale genomic analysis to determine the phylogenetic distribution of the T3SS2 gene cluster and its repertoire of effector proteins. We identified putative T3SS2 gene clusters in 1130 bacterial genomes from 8 bacterial genera, 5 bacterial families and 47 bacterial species. A hierarchical clustering analysis allowed us to define six T3SS2 subgroups (I-VI) with different repertoires of effector proteins, redefining the concepts of T3SS2 core and accessory effector proteins. Finally, we identified a subset of the T3SS2 gene clusters (subgroup VI) that lacks most T3SS2 effector proteins described to date and provided a list of 10 novel effector candidates for this subgroup through bioinformatic analysis. Collectively, our findings indicate that the T3SS2 extends beyond the family Vibrionaceae and suggest that different effector protein repertories could have a differential impact on the pathogenic potential and environmental fitness of each bacterium that has acquired the Vibrio T3SS2 gene cluster.

GrlR, a negative regulator in enteropathogenic E. coli, also represses the expression of LEE virulence genes independently of its interaction with its cognate partner GrlA.

Introduction: Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) belong to a group of pathogens that share the ability to form "attaching and effacing" (A/E) lesions on the intestinal epithelia. A pathogenicity island known as the locus of enterocyte effacement (LEE) contains the genes required for A/E lesion formation. The specific regulation of LEE genes relies on three LEE-encoded regulators: Ler activates the expression of the LEE operons by antagonizing the silencing effect mediated by the global regulator H-NS, GrlA activates ler expression and GrlR represses the expression of the LEE by interacting with GrlA. However, despite the existing knowledge of LEE regulation, the interplay between GrlR and GrlA and their independent roles in gene regulation in A/E pathogens are still not fully understood. Methods: To further explore the role that GrlR and GrlA in the regulation of the LEE, we used different EPEC regulatory mutants and cat transcriptional fusions, and performed protein secretion and expression assays, western blotting and native polyacrylamide gel electrophoresis. Results and discussion: We showed that the transcriptional activity of LEE operons increased under LEE-repressing growth conditions in the absence of GrlR. Interestingly, GrlR overexpression exerted a strong repression effect over LEE genes in wild-type EPEC and, unexpectedly, even in the absence of H-NS, suggesting that GrlR plays an alternative repressor role. Moreover, GrlR repressed the expression of LEE promoters in a non-EPEC background. Experiments with single and double mutants showed that GrlR and H-NS negatively regulate the expression of LEE operons at two cooperative yet independent levels. In addition to the notion that GrlR acts as a repressor by inactivating GrlA through protein-protein interactions, here we showed that a DNA-binding defective GrlA mutant that still interacts with GrlR prevented GrlR-mediated repression, suggesting that GrlA has a dual role as a positive regulator by antagonizing GrlR's alternative repressor role. In line with the importance of the GrlR-GrlA complex in modulating LEE gene expression, we showed that GrlR and GrlA are expressed and interact under both inducing and repressing conditions. Further studies will be required to determine whether the GrlR alternative repressor function depends on its interaction with DNA, RNA, or another protein. These findings provide insight into an alternative regulatory pathway that GrlR employs to function as a negative regulator of LEE genes.

A Brominated Furanone Inhibits Pseudomonas aeruginosa Quorum Sensing and Type III Secretion, Attenuating Its Virulence in a Murine Cutaneous Abscess Model.

Quorum sensing (QS) and type III secretion systems (T3SSs) are among the most attractive anti-virulence targets for combating multidrug-resistant pathogenic bacteria. Some halogenated furanones reduce QS-associated virulence, but their role in T3SS inhibition remains unclear. This study aimed to assess the inhibition of these two systems on Pseudomonas aeruginosa virulence. The halogenated furanones (Z)-4-bromo-5-(bromomethylene)-2(5H) (C-30) and 5-(dibromomethylene)-2(5H) (named hereafter GBr) were synthesized, and their ability to inhibit the secretion of type III exoenzymes and QS-controlled virulence factors was analyzed in P. aeruginosa PA14 and two clinical isolates. Furthermore, their ability to prevent bacterial establishment was determined in a murine cutaneous abscess model. The GBr furanone reduced pyocyanin production, biofilm formation, and swarming motility in the same manner or more effectively than C-30. Moreover, both furanones inhibited the secretion of ExoS, ExoT, or ExoU effectors in all tested strains. The administration of GBr (25 and 50 µM) to CD1 mice infected with the PA14 strain significantly decreased necrosis formation in the inoculation zone and the systemic spread of bacteria more efficiently than C-30 (50 µM). Molecular docking analysis suggested that the gem position of bromine in GBr increases its affinity for the active site of the QS LasR regulator. Overall, our findings showed that the GBr furanone displayed efficient multi-target properties that may favor the development of more effective anti-virulence therapies.

Cyclic di-GMP Regulates the Type III Secretion System and Virulence in Bordetella bronchiseptica.

The second messenger cyclic di-GMP (c-di-GMP) is a ubiquitous molecule in bacteria that regulates diverse phenotypes. Among them, motility and biofilm formation are the most studied. Furthermore, c-di-GMP has been suggested to regulate virulence factors, making it important for pathogenesis. Previously, we reported that c-di-GMP regulates biofilm formation and swimming motility in Bordetella bronchiseptica. Here, we present a multi-omics approach for the study of B. bronchiseptica strains expressing different cytoplasmic c-di-GMP levels, including transcriptome sequencing (RNA-seq) and shotgun proteomics with label-free quantification. We detected 64 proteins significantly up- or downregulated in either low or high c-di-GMP levels and 358 genes differentially expressed between strains with high c-di-GMP levels and the wild-type strain. Among them, we found genes for stress-related proteins, genes for nitrogen metabolism enzymes, phage-related genes, and virulence factor genes. Interestingly, we observed that a virulence factor like the type III secretion system (TTSS) was regulated by c-di-GMP. B. bronchiseptica with high c-di-GMP levels showed significantly lower levels of TTSS components like Bsp22, BopN, and Bcr4. These findings were confirmed by independent methods, such as quantitative reverse transcription-PCR (q-RT-PCR) and Western blotting. Higher intracellular levels of c-di-GMP correlated with an impaired capacity to induce cytotoxicity in a eukaryotic cell in vitro and with attenuated virulence in a murine model. This work presents data that support the role that the second messenger c-di-GMP plays in the pathogenesis of Bordetella.

Comparative transcriptomic analysis provides insights into transcription mechanisms of Vibrio parahaemolyticus T3SS during interaction with HeLa cells.

Vibrio parahaemolyticus is an important foodborne pathogenic bacterium that harbors the type III secretion system 1 (T3SS1) as an essential virulence factor. However, the pathogenesis and infection mechanism mediated by T3SS1 are not entirely clarified. Similar to previous studies on other T3SS-positive bacteria, the T3SS1 needle is a major extracellular component in V. parahaemolyticus. We recently showed that the needle gene-deletion mutant (ΔvscF) exhibited markedly decreased cytotoxicity and effector translocation during interaction with HeLa cells. To further elucidate the pathogenesis of T3SS1 during host cell infection, bacterial RNA was extracted from wild-type POR-1 and ΔvscF mutants under infected condition for comparative RNA sequencing analysis in HeLa cell. The results showed that 120 differentially expressed genes (DEGs) were identified in the ΔvscF-infected group. These encoded proteins of DEGs, such as VP2088, VP2089, and VP2091, were annotated as ABC transporter system, whereas VP0757, VP1123, and VP1289 may be new transcriptional regulators. In addition, the downregulation of T3SS1 had a positive influence on the expression of T3SS2. Moreover, the transcription of the basal body is unaffected by the needle, and there was a close relation among the tip, translocon, and needle, because bacterial adenylate cyclase two-hybrid system (BACTH system) assay indicated the interaction of VP1656, VP1670, VP1693, and VP1694 (VscF). This study provides insights into transcription mechanism of T3SS1 upon infecting HeLa cell, which is expected to better clarify the T3SS1 virulent mechanism.

Anti-Virulence Properties of Plant Species: Correlation between In Vitro Activity and Efficacy in a Murine Model of Bacterial Infection.

Several plant extracts exhibit anti-virulence properties due to the interruption of bacterial quorum sensing (QS). However, studies on their effects at the preclinical level are scarce. Here, we used a murine model of abscess/necrosis induced by Pseudomonas aeruginosa to evaluate the anti-pathogenic efficacy of 24 plant extracts at a sub-inhibitory concentration. We analyzed their ability to inhibit QS-regulated virulence factors such as swarming, pyocyanin production, and secretion of the ExoU toxin via the type III secretion system (T3SS). Five of the seven extracts with the best anti-pathogenic activity reduced ExoU secretion, and the extracts of Diphysa americana and Hibiscus sabdariffa were identified as the most active. Therefore, the abscess/necrosis model allows identification of plant extracts that have the capacity to reduce pathogenicity of P. aeruginosa. Furthermore, we evaluated the activity of the plant extracts on Chromobacterium violaceum. T3SS (ΔescU) and QS (ΔcviI) mutant strains were assessed in both the abscess/necrosis and sepsis models. Only the ΔescU strain had lower pathogenicity in the animal models, although no activity of plant extracts was observed. These results demonstrate differences between the anti-virulence activity recorded in vitro and pathogenicity in vivo and between the roles of QS and T3S systems as virulence determinants.

Anti-Pathogenic Properties of the Combination of a T3SS Inhibitory Halogenated Pyrrolidone with C-30 Furanone.

Antimicrobial resistance is one of the current public health challenges to be solved. The World Health Organization (WHO) has urgently called for the development of strategies to expand the increasingly limited antimicrobial arsenal. The development of anti-virulence therapies is a viable option to counteract bacterial infections with the possibility of reducing the generation of resistance. Here we report on the chemical structures of pyrrolidones DEXT 1-4 (previously identified as furan derivatives) and their anti-virulence activity on Pseudomonas aeruginosa strains. DEXT 1-4 were shown to inhibit biofilm formation, swarming motility, and secretion of ExoU and ExoT effector proteins. Also, the anti-pathogenic property of DEXT-3 alone or in combination with furanone C-30 (quorum sensing inhibitor) or MBX-1641 (type III secretion system inhibitor) was analyzed in a model of necrosis induced by P. aeruginosa PA14. All treatments reduced necrosis; however, only the combination of C-30 50 µM with DEXT-3 100 µM showed significant inhibition of bacterial growth in the inoculation area and systemic dispersion. In conclusion, pyrrolidones DEXT 1-4 are chemical structures capable of reducing the pathogenicity of P. aeruginosa and with the potential for the development of anti-virulence combination therapies.

Identification of a Family of Vibrio Type III Secretion System Effectors That Contain a Conserved Serine/Threonine Kinase Domain.

Vibrio parahaemolyticus is a marine Gram-negative bacterium that is a leading cause of seafood-borne gastroenteritis. Pandemic strains of V. parahaemolyticus rely on a specialized protein secretion machinery known as the type III secretion system 2 (T3SS2) to cause disease. The T3SS2 mediates the delivery of effector proteins into the cytosol of infected cells, where they subvert multiple cellular pathways. Here, we identify a new T3SS2 effector protein encoded by VPA1328 (VP_RS21530) in V. parahaemolyticus RIMD2210633. Bioinformatic analysis revealed that VPA1328 is part of a larger family of uncharacterized T3SS effector proteins with homology to the VopG effector protein in Vibrio cholerae AM-19226. These VopG-like proteins are found in many but not all T3SS2 gene clusters and are distributed among diverse Vibrio species, including V. parahaemolyticus, V. cholerae, V. mimicus, and V. diabolicus and also in Shewanella baltica. Structure-based prediction analyses uncovered the presence of a conserved C-terminal kinase domain in VopG orthologs, similar to the serine/threonine kinase domain found in the NleH family of T3SS effector proteins. However, in contrast to NleH effector proteins, in tissue culture-based infections, VopG did not impede host cell death or suppress interleukin 8 (IL-8) secretion, suggesting a yet undefined role for VopG during V. parahaemolyticus infection. Collectively, our work reveals that VopG effector proteins, a new family of likely serine/threonine kinases, is widely distributed in the T3SS2 effector armamentarium among marine bacteria. IMPORTANCE Vibrio parahaemolyticus is the leading bacterial cause of seafood-borne gastroenteritis worldwide. The pathogen relies on a type III secretion system to deliver a variety of effector proteins into the cytosol of infected cells to subvert cellular function. In this study, we identified a novel Vibrio parahaemolyticus effector protein that is similar to the VopG effector of Vibrio cholerae. VopG-like effectors were found in diverse Vibrio species and contain a conserved serine/threonine kinase domain that bears similarity to the kinase domain in the enterohemorrhagic Escherichia coli (EHEC) and Shigella NleH effectors that manipulate host cell survival pathways and host immune responses. Together our findings identify a new family of Vibrio effector proteins and highlight the role of horizontal gene transfer events among marine bacteria in shaping T3SS gene clusters.

CesL Regulates Type III Secretion Substrate Specificity of the Enteropathogenic E. coli Injectisome.

The type III secretion system (T3SS) is a complex molecular device used by several pathogenic bacteria to translocate effector proteins directly into eukaryotic host cells. One remarkable feature of the T3SS is its ability to secrete different categories of proteins in a hierarchical manner, to ensure proper assembly and timely delivery of effectors into target cells. In enteropathogenic Escherichia coli, the substrate specificity switch from translocator to effector secretion is regulated by a gatekeeper complex composed of SepL, SepD, and CesL proteins. Here, we report a characterization of the CesL protein using biochemical and genetic approaches. We investigated discrepancies in the phenotype among different cesL deletion mutants and showed that CesL is indeed essential for translocator secretion and to prevent premature effector secretion. We also demonstrated that CesL engages in pairwise interactions with both SepL and SepD. Furthermore, while association of SepL to the membrane does not depended on CesL, the absence of any of the proteins forming the heterotrimeric complex compromised the intracellular stability of each component. In addition, we found that CesL interacts with the cytoplasmic domains of the export gate components EscU and EscV. We propose a mechanism for substrate secretion regulation governed by the SepL/SepD/CesL complex.

High mortality by nosocomial infections caused by carbapenem-resistant P. aeruginosa in a referral hospital in Brazil: facing the perfect storm.

Introduction. Carbapenem-resistant Pseudomonas aeruginosa is responsible for increased patient mortality.Gap Statement. Five and 30 day in-hospital all-cause mortality in patients with P. aeruginosa infections were assessed, followed by evaluations concerning potential correlations between the type III secretion system (TTSS) genotype and the production of metallo-ß-lactamase (MBL).Methodology. This assessment comprised a retrospective cohort study including consecutive patients with carbapenem-resistant infections hospitalized in Brazil from January 2009 to June 2019. PCR analyses were performed to determine the presence of TTSS-encoding genes and MBL genes.Results. The 30-day and 5-day mortality rates for 262 patients were 36.6 and 17.9 %, respectively. The unadjusted survival probabilities for up to 5 days were 70.55 % for patients presenting exoU-positive isolates and 86 % for those presenting exo-negative isolates. The use of urinary catheters, as well as the presence of comorbidity conditions, secondary bacteremia related to the respiratory tract, were independently associated with death at 5 and 30 days. The exoS gene was detected in 64.8 % of the isolates, the presence of the exoT and exoY genes varied and exoU genes occurred in 19.3 % of the isolates. The exoU genotype was significantly more frequent among multiresistant strains. MBL genes were not detected in 92 % of the isolates.Conclusions. Inappropriate therapy is a crucial factor regarding the worse prognosis among patients with infections caused by multiresistant P. aeruginosa, especially those who died within 5 days of diagnosis, regardless of the genotype associated with TTSS virulence.

Genomic characterization of the non-O1/non-O139 Vibrio cholerae strain that caused a gastroenteritis outbreak in Santiago, Chile, 2018.

Vibrio cholerae is a human pathogen, which is transmitted by the consumption of contaminated food or water. V. cholerae strains belonging to the serogroups O1 and O139 can cause cholera outbreaks and epidemics, a severe life-threatening diarrheal disease. In contrast, serogroups other than O1 and O139, denominated as non-O1/non-O139, have been mainly associated with sporadic cases of moderate or mild diarrhea, bacteremia and wound infections. Here we investigated the virulence determinants and phylogenetic origin of a non-O1/non-O139 V. cholerae strain that caused a gastroenteritis outbreak in Santiago, Chile, 2018. We found that this outbreak strain lacks the classical virulence genes harboured by O1 and O139 strains, including the cholera toxin (CT) and the toxin-coregulated pilus (TCP). However, this strain carries genomic islands (GIs) encoding Type III and Type VI secretion systems (T3SS/T6SS) and antibiotic resistance genes. Moreover, we found these GIs are wide distributed among several lineages of non-O1/non-O139 strains. Our results suggest that the acquisition of these GIs may enhance the virulence of non-O1/non-O139 strains that lack the CT and TCP-encoding genes. Our results highlight the pathogenic potential of these V. cholerae strains.

Horizontally Acquired Quorum-Sensing Regulators Recruited by the PhoP Regulatory Network Expand the Host Adaptation Repertoire in the Phytopathogen Pectobacterium brasiliense.

In this study, we examine the impact of transcriptional network rearrangements driven by horizontal gene acquisition in PhoP and SlyA regulons using as a case study a phytopathosystem comprised of potato tubers and the soft-rot pathogen Pectobacterium brasiliense 1692 (Pb1692). Genome simulations and statistical analyses uncovered the tendency of PhoP and SlyA networks to mobilize lineage-specific traits predicted as horizontal gene transfer at late infection, highlighting the prominence of regulatory network rearrangements in this stage of infection. The evidence further supports the circumscription of two horizontally acquired quorum-sensing regulators (carR and expR1) by the PhoP network. By recruiting carR and expR1, the PhoP network also impacts certain host adaptation- and bacterial competition-related systems, seemingly in a quorum sensing-dependent manner, such as the type VI secretion system, carbapenem biosynthesis, and plant cell wall-degrading enzymes (PCWDE) like cellulases and pectate lyases. Conversely, polygalacturonases and the type III secretion system (T3SS) exhibit a transcriptional pattern that suggests quorum-sensing-independent regulation by the PhoP network. This includes an uncharacterized novel phage-related gene family within the T3SS gene cluster that has been recently acquired by two Pectobacterium species. The evidence further suggests a PhoP-dependent regulation of carbapenem- and PCWDE-encoding genes based on the synthesized products' optimum pH. The PhoP network also controls slyA expression in planta, which seems to impact carbohydrate metabolism regulation, especially at early infection, when 76.2% of the SlyA-regulated genes from that category also require PhoP to achieve normal expression levels.IMPORTANCE Exchanging genetic material through horizontal transfer is a critical mechanism that drives bacteria to efficiently adapt to host defenses. In this report, we demonstrate that a specific plant-pathogenic species (from the Pectobacterium genus) successfully integrated a population density-based behavior system (quorum sensing) acquired through horizontal transfer into a resident stress-response gene regulatory network controlled by the PhoP protein. Evidence found here underscores that subsets of bacterial weaponry critical for colonization, typically known to respond to quorum sensing, are also controlled by PhoP. Some of these traits include different types of enzymes that can efficiently break down plant cell walls depending on the environmental acidity level. Thus, we hypothesize that PhoP's ability to elicit regulatory responses based on acidity and nutrient availability fluctuations has strongly impacted the fixation of its regulatory connection with quorum sensing. In addition, another global gene regulator, known as SlyA, was found under the PhoP regulatory network. The SlyA regulator controls a series of carbohydrate metabolism-related traits, which also seem to be regulated by PhoP. By centralizing quorum sensing and slyA under PhoP scrutiny, Pectobacterium cells added an advantageous layer of control over those two networks that potentially enhances colonization efficiency.

Secretory immunoglobulin A (sIgA) in saliva versus virulence proteins of enteropathogenic Escherichia coli (EPEC) in ill and colonized children.

INTRODUCTION: We evaluated the presence of sIgA in saliva, versus Escherichia coli secreted proteins (Esp) related to the type III secretion system (T3SS), and its semi-quantitative concentration in children under 2 years-old (no longer breastfed) who were previously colonized or infected with enteropathogenic E. coli (EPEC). METHODS: We analyzed the presence of sIgA in 40 children, who previously had positive cultures for EPEC associated (n=17) or not associated (n=23) with diarrhea, using the Western Blot technique versus E. coli secreted proteins: EspABCD. A semi-quantitative measurement of the reaction for each protein was made by its density peaks (OD). RESULTS: We found sIgA versus all or some EspABCD proteins in both groups. However, the ill patients had higher concentrations of these antibodies than colonized patients. DISCUSSION: The presence of sIgA in saliva could reflect an intestinal immune response and their levels could be related to a greater exposure and/or bacterial load.

Tetradecanoic Acids With Anti-Virulence Properties Increase the Pathogenicity of Pseudomonas aeruginosa in a Murine Cutaneous Infection Model.

Blocking virulence is a promising alternative to counteract Pseudomonas aeruginosa infections. In this regard, the phenomenon of cell-cell communication by quorum sensing (QS) is an important anti-virulence target. In this field, fatty acids (FA) have gained notoriety for their role as autoinducers, as well as anti-virulence molecules in vitro, like some saturated FA (SAFA). In this study, we analyzed the anti-virulence activity of SAFA with 12 to18 carbon atoms and compared their effect with the putative autoinducer cis-2-decenoic acid (CDA). The effect of SAFA on six QS-regulated virulence factors and on the secretion of the exoenzyme ExoU was evaluated. In addition, a murine cutaneous infection model was used to determine their influence on the establishment and damage caused by P. aeruginosa PA14. Dodecanoic (lauric, C12:0) and tetradecanoic (myristic, C14:0) acids (SAFA C12-14) reduced the production of pyocyanin by 35-58% at 40 and 1,000 µM, while CDA inhibited it 62% at a 3.1 µM concentration. Moreover, the SAFA C12-14 reduced swarming by 90% without affecting biofilm formation. In contrast, CDA reduced the biofilm by 57% at 3 µM but did not affect swarming. Furthermore, lauric and myristic acids abolished ExoU secretion at 100 and 50 µM respectively, while CDA reduced it by ≈ 92% at 100 µM. Remarkably, the coadministration of myristic acid (200 and 1,000 µM) with P. aeruginosa PA14 induced greater damage and reduced survival of the animals up to 50%, whereas CDA to 500 µM reduced the damage without affecting the viability of the PA14 strain. Hence, our results show that SAFA C12-14 and CDA have a role in regulation of P. aeruginosa virulence, although their inhibition/activation molecular mechanisms are different in complex environments such as in vivo systems.

Gene expression analysis identifies hypothetical genes that may be critical during the infection process of Xanthomonas citri subsp. citri

Background: Gene expression analysis via microarray is widely used in phytobacteria to validate differential gene expression associated with virulence or to compare biological profiles of wild type and mutant strains. Here, we employed DNA microarrays to study the early stages of the infection process (24, 72 and 120 h post-inoculation) of Xanthomonas citri subsp. citri (Xac) infecting Citrus sinensis to interrogate the expression profiles of hypothetical genes. Results: Under infective conditions, 446 genes were up- and 306 downregulated. Outstanding among genes upregulated during infection were those involved in synthesizing the Type 3 Secretion System and effectors, xanthan gum and quorum-sensing induction, and flagellum synthesis and regulation. Additionally, 161 hypothetical genes were up- and 100 were downregulated, 49 of which are known to have a significant biological role. To understand hypothetical gene co-regulation or -expression, nine expression profiles including 158 genes were identified during the three infection phases. Of these, 47 hypothetical genes were identified as having expression profiles associated with at least one connected to a gene associated with adaptation and virulence. Conclusions: Expression patterns of six differentially expressed genes were validated by quantitative reverse transcription polymerase chain reaction, thus demonstrating the effectiveness of this tool in global gene expression analysis in Xac.

The Type III Secretion System (T3SS)-Translocon of Atypical Enteropathogenic Escherichia coli (aEPEC) Can Mediate Adherence.

The intimin protein is the major adhesin involved in the intimate adherence of atypical enteropathogenic Escherichia coli (aEPEC) strains to epithelial cells, but little is known about the structures involved in their early colonization process. A previous study demonstrated that the type III secretion system (T3SS) plays an additional role in the adherence of an Escherichia albertii strain. Therefore, we assumed that the T3SS could be related to the adherence efficiency of aEPEC during the first stages of contact with epithelial cells. To test this hypothesis, we examined the adherence of seven aEPEC strains and their eae (intimin) isogenic mutants in the standard HeLa adherence assay and observed that all wild-type strains were adherent while five isogenic eae mutants were not. The two eae mutant strains that remained adherent were then used to generate the eae/escN double mutants (encoding intimin and the T3SS ATPase, respectively) and after the adherence assay, we observed that one strain lost its adherence capacity. This suggested a role for the T3SS in the initial adherence steps of this strain. In addition, we demonstrated that this strain expressed the T3SS at significantly higher levels when compared to the other wild-type strains and that it produced longer translocon-filaments. Our findings reveal that the T3SS-translocon can play an additional role as an adhesin at the beginning of the colonization process of aEPEC.

The type III secretion system (T3SS)-Translocon of atypical enteropathogenic Escherichia coli (aEPEC) can mediate adherence

The intimin protein is the major adhesin involved in the intimate adherence of atypicalenteropathogenicEscherichia coli(aEPEC) strains to epithelial cells, but little is knownabout the structures involved in their early colonization process. A previous studydemonstrated that the type III secretion system (T3SS) plays an additional role in theadherence of anEscherichia albertiistrain. Therefore, we assumed that the T3SS couldbe related to the adherence efficiency of aEPEC during the first stages of contactwith epithelial cells. To test this hypothesis, we examined the adherence of sevenaEPEC strains and theireae(intimin) isogenic mutants in the standard HeLa adherenceassay and observed that all wild-type strains were adherent while five isogeniceaemutants were not. The twoeaemutant strains that remained adherent were then usedto generate theeae/escNdouble mutants (encoding intimin and the T3SS ATPase,respectively) and after the adherence assay, we observed that one strain lost itsadherence capacity. This suggested a role for the T3SS in the initial adherence stepsof this strain. In addition, we demonstrated that this strain expressed the T3SS atsignificantly higher levels when compared to the other wild-type strains and that itproduced longer translocon-filaments. Our findings reveal that the T3SS-transloconcan play an additional role as an adhesin at the beginning of the colonization processof aEPEC.

Novel Effector Protein EspY3 of Type III Secretion System from Enterohemorrhagic Escherichia coli Is Localized in Actin Pedestals.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic Escherichia coli (EPEC) are attaching and effacing (A/E) pathogens, which translocate effector proteins to intestinal enterocytes through a type III secretion system (T3SS). T3SS and most of its effector proteins are encoded in a pathogenicity island called LEE. Recently, new effectors have been located outside the LEE. This study aimed to characterize EspY3, a novel non-LEE encoded T3SS effector of EHEC. EspY3 shares homology with SopD and PipB2 effector proteins of Salmonella's T3SS-1 and T3SS-2, respectively. The presence of recombinant EspY3 in the supernatant samples demonstrated that EspY3 was secreted by the T3SS of EHEC and EPEC. Through infection assays, we demonstrated the translocation of EspY3 into Caco-2 cells by T3SS of EPEC. The subcellular localization of EspY3 was determined in the pedestal region, where its presence generates a significant increase in the size of the pedestals area. The EspY3 effector induced the elongation of polymerized actin pedestals in infected Caco-2 by EPEC. This study confirmed that EspY3 is part of the repertoire of T3SS effectors of EHEC O157:H7, and that it participates in modeling cellular actin during the infection.