Pseudomonas aeruginosa Isolates from Water Samples of the Gulf of Mexico Show Similar Virulence Properties but Different Antibiotic Susceptibility Profiles than Clinical Isolates.

Pseudomonas aeruginosa is an opportunistic pathogen found in a wide variety of environments, including soil, water, and habitats associated with animals, humans, and plants. From a One Health perspective, which recognizes the interconnectedness of human, animal, and environmental health, it is important to study the virulence characteristics and antibiotic susceptibility of environmental bacteria. In this study, we compared the virulence properties and the antibiotic resistance profiles of seven isolates collected from the Gulf of Mexico with those of seven clinical strains of P. aeruginosa. Our results indicate that the marine and clinical isolates tested exhibit similar virulence properties; they expressed different virulence factors and were able to kill Galleria mellonella larvae, an animal model commonly used to analyze the pathogenicity of many bacteria, including P. aeruginosa. In contrast, the clinical strains showed higher antibiotic resistance than the marine isolates. Consistently, the clinical strains exhibited a higher prevalence of class 1 integron, an indicator of anthropogenic impact, compared with the marine isolates. Thus, our results indicate that the P. aeruginosa marine strains analyzed in this study, isolated from the Gulf of Mexico, have similar virulence properties, but lower antibiotic resistance, than those from hospitals.

Interactions between the AraC/XylS-like transcriptional activator InvF of Salmonella Typhimurium, the RNA polymerase alpha subunit and the chaperone SicA.

Salmonella enterica serovar Typhimurium causes gastroenteritis and systemic infections in humans. For this bacterium the expression of a type III secretion system (T3SS) and effector proteins encoded in the Salmonella pathogenicity island-1 (SPI-1), is keystone for the virulence of this bacterium. Expression of these is controlled by a regulatory cascade starting with the transcriptional regulators HilD, HilC and RtsA that induce the expression of HilA, which then activates expression of the regulator InvF, a transcriptional regulator of the AraC/XylS family. InvF needs to interact with the chaperone SicA to activate transcription of SPI-1 genes including sicA, sopB, sptP, sopE, sopE2, and STM1239. InvF very likely acts as a classical activator; however, whether InvF interacts with the RNA polymerase alpha subunit RpoA has not been determined. Results from this study confirm the interaction between InvF with SicA and reveal that both proteins interact with the RNAP alpha subunit. Thus, our study further supports that the InvF/SicA complex acts as a classical activator. Additionally, we showed for the first time an interaction between a chaperone of T3SS effectors (SicA) and the RNAP.

Genomic and phenotypic characterization of Pseudomonas sp. GOM7, a novel marine bacterial species with antimicrobial activity against multidrug-resistant Staphylococcus aureus.

Antimicrobial resistance (AMR) represents a serious threat to global health. The development of new drugs to combat infections caused by bacteria resistant to multiple or even all available antibiotics is urgent. Most antibiotics used up to date have been identified from soil microorganisms. The marine environment represents an alternative source with great potential for the identification of microorganisms that produce bioactive molecules, including antibiotics. In this study, we analyzed the antibacterial activity of a collection of 82 bacterial strains isolated from marine water and sediment samples collected from the Southwestern Gulf of Mexico. Eight of the marine isolates inhibited the growth of different pathogenic bacteria, seven of which were identified as presumptive Pseudomonas aeruginosa. Interestingly, genome sequencing and phylogenetic analysis revealed that the remaining marine isolate showing antibacterial activity is a novel Pseudomonas species that we denominated Pseudomonas sp. GOM7, which was not pathogenic in the Galleria mellonella infection model in the conditions tested. Notably, Pseudomonas sp. GOM7 inhibited the growth of multidrug and methicillin-resistant strains of the priority pathogen Staphylococcus aureus. Our results show that the anti-S. aureus compound(s) produced by Pseudomonas sp. GOM7 can be extracted from the culture supernatant of this bacterium with the organic solvent ethyl acetate. Annotation of the Pseudomonas sp. GOM7 genome revealed the presence of several biosynthetic gene clusters predicted to code for possible antimicrobial compounds. Our results further highlight the potential of bacteria from the Gulf of Mexico as a source of novel antimicrobials.

Proteomic analysis reveals the global effect of the BarA/SirA-Csr regulatory cascade in Salmonella Typhimurium grown in conditions that favor the expression of invasion genes.

In many bacteria, the BarA/SirA and Csr regulatory systems control expression of genes encoding a wide variety of cellular functions. The BarA/SirA two-component system induces the expression of CsrB and CsrC, two small non-coding RNAs that sequester CsrA, a protein that binds to target mRNAs and thus negatively or positively regulates their expression. BarA/SirA and CsrB/C induce expression of the Salmonella Pathogenicity Island 1 (SPI-1) genes required for Salmonella invasion of host cells. To further investigate the regulatory role of the BarA/SirA and Csr systems in Salmonella, we performed LC-MS/MS proteomic analysis using the WT S. Typhimurium strain and its derived ΔsirA and ΔcsrB ΔcsrC mutants grown in SPI-1-inducing conditions. The expression of 164 proteins with a wide diversity, or unknown, functions was significantly affected positively or negatively by the absence of SirA and/or CsrB/C. Interestingly, 19 proteins were identified as new targets for SirA-CsrB/C. Our results support that SirA and CsrB/C act in a cascade fashion to regulate gene expression in S. Typhimurium in the conditions tested. Notably, our results show that SirA-CsrB/C-CsrA controls expression of proteins required for the replication of Salmonella in the intestinal lumen, in an opposite way to its control exerted on the SPI-1 proteins. SIGNIFICANCE: The BarA/SirA and Csr global regulatory systems control a wide range of cellular processes, including the expression of virulence genes. For instance, in Salmonella, BarA/SirA and CsrB/C positively regulate expression of the SPI-1 genes, which are required for Salmonella invasion to host cells. In this study, by performing a proteomic analysis, we identified 164 proteins whose expression was positively or negatively controlled by SirA and CsrB/C in SPI-1-inducing conditions, including 19 new possible targets of these systems. Our results support the action of SirA and CsrB/C in a cascade fashion to control different cellular processes in Salmonella. Interestingly, our data indicate that SirA-CsrB/C-CsrA controls inversely the expression of proteins required for invasion of the intestinal epithelium and for replication in the intestinal lumen, which suggests a role for this regulatory cascade as a molecular switch for Salmonella virulence. Thus, our study further expands the insight into the regulatory mechanisms governing the virulence and physiology of an important pathogen.

Antibacterial and antibiofilm activities of ZIF-67.

Currently, antibiotic-resistant bacteria represent a serious threat to public health worldwide. Biofilm formation potentiates both virulence and antibiotic resistance of bacteria. Therefore, the discovery of new antibacterial and antibiofilm compounds is an issue of paramount importance to combat and prevent hard-to-treat bacterial infections. Zeolitic-imidazolate-frameworks (ZIFs) are metallo-organic compounds known to have various interesting chemical and biological applications, including antibacterial properties. In this study, we synthesized ZIF-67 nanoparticles, formed by imidazolate anions and cobalt cations, and found that they inhibit the growth of Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus. Sub-inhibitory concentrations of ZIF-67 were also able to significantly reduce the biomass of pre-established biofilms of these pathogenic bacteria. On the other hand, the ZIF-67 nanoparticles had null or low cytotoxicity in mammalian cells at those concentrations showing antibacterial or antibiofilm activities. Thus, our results reveal the potential of ZIF-67 nanoparticles to be used against pathogenic bacteria.

CsrA Positively and Directly Regulates the Expression of the pdu, pocR, and eut Genes Required for the Luminal Replication of Salmonella Typhimurium.

Enteric pathogens, such as Salmonella, have evolved to thrive in the inflamed gut. Genes located within the Salmonella pathogenicity island 1 (SPI-1) mediate the invasion of cells from the intestinal epithelium and the induction of an intestinal inflammatory response. Alternative electron acceptors become available in the inflamed gut and are utilized by Salmonella for luminal replication through the metabolism of propanediol and ethanolamine, using the enzymes encoded by the pdu and eut genes. The RNA-binding protein CsrA inhibits the expression of HilD, which is the central transcriptional regulator of the SPI-1 genes. Previous studies suggest that CsrA also regulates the expression of the pdu and eut genes, but the mechanism for this regulation is unknown. In this work, we show that CsrA positively regulates the pdu genes by binding to the pocR and pduA transcripts as well as the eut genes by binding to the eutS transcript. Furthermore, our results show that the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the pdu and eut genes mediated by PocR or EutR, which are the positive AraC-like transcriptional regulators for the pdu and eut genes, respectively. By oppositely regulating the expression of genes for invasion and for luminal replication, the SirA-CsrB/CsrC-CsrA regulatory cascade could be involved in the generation of two Salmonella populations that cooperate for intestinal colonization and transmission. Our study provides new insight into the regulatory mechanisms that govern Salmonella virulence. IMPORTANCE The regulatory mechanisms that control the expression of virulence genes are essential for bacteria to infect hosts. Salmonella has developed diverse regulatory mechanisms to colonize the host gut. For instance, the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the SPI-1 genes, which are required for this bacterium to invade intestinal epithelium cells and for the induction of an intestinal inflammatory response. In this study, we determine the mechanisms by which the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the pdu and eut genes, which are necessary for the replication of Salmonella in the intestinal lumen. Thus, our data, together with the results of previous reports, indicate that the SirA-CsrB/CsrC-CsrA regulatory cascade has an important role in the intestinal colonization by Salmonella.

hilD Is Required for the Active Internalization of Salmonella Newport into Cherry Tomatoes.

Salmonella enterica is a foodborne pathogen that can be internalized into fresh produce. Most of the Salmonella virulence genes are clustered in regions denominated Salmonella Pathogenicity Islands (SPI). SPI-1 encodes a Type Three Secretion System (T3SS-1) and effector proteins that allow the internalization of Salmonella into animal cells. HilD is a transcriptional regulator that induces the expression of SPI-1 genes and other related virulence genes located outside of this island. Here, we assessed the role of hilD in the internalization of Salmonella Newport and Typhimurium into cherry tomatoes, by evaluating either an isolate from an avocado orchard, S. Newport-45 or the laboratory strain S. Typhimurium SL1344 and their isogenic mutants in hilD. The internalization of these bacteria was carried out by using a temperature gradient of 12°C. The transcription of hilD and invA was tested by qRT-PCR experiments. Our results show that S. Newport-45 hilD mutant viable cells obtained from the interior of the fruit were decreased (2.7-fold), compared with those observed for S. Typhimurium SL1344. Interestingly, at 3 days postinoculation, the cells recovered from S. Newport-45 hilD mutant were similar to those recovered from all the strains evaluated, suggesting that hilD is required only for the initial internalization of S. Newport.

Inducing Self-Poisoning: a Feasible Antibacterial Strategy?

Development of novel antibacterial strategies is required to tackle the alarming threat for global health due to antimicrobial resistance. In this issue of the Journal of Bacteriology, Boulanger et al. provide evidence supporting that the blocking of metabolic pathways to induce accumulation of toxic intermediates can be a possible approach to combat bacterial infections (E. F. Boulanger, A. Sabag-Daigle, M. Baniasad, K. Kokkinias, et al., J Bacteriol 204:e00344-22, 2022, https://doi.org/10.1128/jb.00344-22).

Two Additional Connections between the Transcriptional Programs Controlling Invasion and Intracellular Replication of Salmonella: HilD-SprB Positively Regulates phoP and slyA.

Salmonella virulence relies on the ability of this bacterium to invade the intestinal epithelium and to replicate inside macrophages, which are functions mainly encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), respectively. Complex regulatory programs control the expression of SPI-1 and SPI-2 and functionally related genes, involving the integration of ancestral regulators and regulators that Salmonella has acquired during its evolution. Interestingly, some previous studies have revealed cross talk between the regulatory programs for SPI-1 and SPI-2. Here, we report two additional connections between the regulatory programs controlling the expression of genes for invasion and intracellular replication. Our results show that the acquired regulators HilD and SprB, both encoded in SPI-1, induce, in a cascade fashion, the expression of PhoP and SlyA, two ancestral regulators that activate the expression of SPI-2 and other genes required for intracellular replication. We provide evidence supporting that the regulation of phoP and slyA by HilD-SprB was adapted during the divergence of Salmonella from its closer species, Escherichia coli, with the acquisition of SPI-1 and thus the gain of HilD and SprB, as well as through cis-regulatory evolution of phoP and slyA. Therefore, our study further expands the knowledge about the intricate regulatory network controlling the expression of virulence genes in Salmonella. IMPORTANCE Bacteria have developed diverse regulatory mechanisms to control genetic expression, in the case of pathogenic bacteria, to induce the expression of virulence genes in particular niches during host infection. In Salmonella, an intricate regulatory network has been determined, which controls the spatiotemporal expression of the SPI-1 and SPI-2 gene clusters that mediate the invasion to and the replication inside host cells, respectively. In this study, we report two additional pathways of cross talk between the transcriptional programs for SPI-1 and SPI-2. Additionally, our results support that these additional regulatory pathways were adapted during the divergence of Salmonella from its closer species, Escherichia coli. This study further expands the knowledge about the mechanisms determining the Salmonella virulence.

The Two-Component System CpxRA Represses Salmonella Pathogenicity Island 2 by Directly Acting on the ssrAB Regulatory Operon.

The acquisition of Salmonella pathogenicity island 2 (SPI-2) conferred on Salmonella the ability to survive and replicate within host cells. The ssrAB bicistronic operon, located in SPI-2, encodes the SsrAB two-component system (TCS), which is the central positive regulator that induces the expression of SPI-2 genes as well as other genes located outside this island. On the other hand, CpxRA is a two-component system that regulates expression of virulence genes in many bacteria in response to different stimuli that perturb the cell envelope. We previously reported that the CpxRA system represses the expression of SPI-1 and SPI-2 genes under SPI-1-inducing conditions by decreasing the stability of the SPI-1 regulator HilD. Here, we show that under SPI-2-inducing conditions, which mimic the intracellular environment, CpxRA represses the expression of SPI-2 genes by the direct action of phosphorylated CpxR (CpxR-P) on the ssrAB regulatory operon. CpxR-P recognized two sites located proximal and distal from the promoter located upstream of ssrA. Consistently, we found that CpxRA reduces the replication of Salmonella enterica serovar Typhimurium inside murine macrophages. Therefore, our results reveal CpxRA as an additional regulator involved in the intracellular lifestyle of Salmonella, which in turn adds a new layer to the intricate regulatory network controlling the expression of Salmonella virulence genes. IMPORTANCE SPI-2 encodes a type III secretion system (T3SS) that is a hallmark for the species Salmonella enterica, which is essential for the survival and replication within macrophages. Expression of SPI-2 genes is positively controlled by the two-component system SsrAB. Here, we determined a regulatory mechanism involved in controlling the overgrowth of Salmonella inside macrophages. In this mechanism, CpxRA, a two-component system that is activated by extracytoplasmic stress, directly represses expression of the ssrAB regulatory operon; as a consequence, expression of SsrAB target genes is decreased. Our findings reveal a novel mechanism involved in the intracellular lifestyle of Salmonella, which is expected to sense perturbations in the bacterial envelope that Salmonella faces inside host cells, as the synthesis of the T3SS-2 itself.

Regulatory Evolution of the phoH Ancestral Gene in Salmonella enterica Serovar Typhimurium.

One important event for the divergence of Salmonella from Escherichia coli was the acquisition by horizontal transfer of the Salmonella pathogenicity island 1 (SPI-1), containing genes required for the invasion of host cells by Salmonella. HilD is an AraC-like transcriptional regulator in SPI-1 that induces the expression of the SPI-1 and many other acquired virulence genes located in other genomic regions of Salmonella. Additionally, HilD has been shown to positively control the expression of some ancestral genes (also present in E. coli and other bacteria), including phoH. In this study, we determined that both the gain of HilD and cis-regulatory evolution led to the integration of the phoH gene into the HilD regulon. Our results indicate that a HilD-binding sequence was generated in the regulatory region of the S. enterica serovar Typhimurium phoH gene, which mediates the activation of promoter 1 of this gene under SPI-1-inducing conditions. Furthermore, we found that repression by H-NS, a histone-like protein, was also adapted on the S. Typhimurium phoH gene and that HilD activates the expression of this gene in part by antagonizing H-NS. Additionally, our results revealed that the expression of the S. Typhmurium phoH gene is also activated in response to low phosphate but independently of the PhoB/R two-component system, known to regulate the E. coli phoH gene in response to low phosphate. Thus, our results indicate that cis-regulatory evolution has played a role in the expansion of the HilD regulon and illustrate the phenomenon of differential regulation of ortholog genes. IMPORTANCE Two mechanisms mediating differentiation of bacteria are well known: acquisition of genes by horizontal transfer events and mutations in coding DNA sequences. In this study, we found that the phoH ancestral gene is differentially regulated between Salmonella Typhimurium and Escherichia coli, two closely related bacterial species. Our results indicate that this differential regulation was generated by mutations in the regulatory sequence of the S. Typhimurium phoH gene and by the acquisition by S. Typhimurium of foreign DNA encoding the transcriptional regulator HilD. Thus, our results, together with those from an increasing number of studies, indicate that cis-regulatory evolution can lead to the rewiring and reprogramming of transcriptional regulation, which also plays an important role in the divergence of bacteria through time.

Long-chain fatty acids alter transcription of Helicobacter pylori virulence and regulatory genes.

Infection with Helicobacter pylori is one of the most important risk factors for developing gastric cancer (GC). The type IV secretion system (T4SS) encoded in the cag pathogenicity island is the main virulence factor of H. pylori associated with GC. Additionally, other virulence factors have been shown to play a role in the H. pylori virulence, such as vacuolizing cytotoxin (VacA), urease, flagella, and adhesins. Long-chain fatty acids (LCFAs) are signaling molecules that affect the transcription of virulence genes in several pathogenic bacteria such as Salmonella enterica, Vibrio cholerae, Pseudomonas aeruginosa and Mycobacterium tuberculosis. However, the effect of LCFAs on the transcription of H. pylori virulence and regulatory genes remains unknown. Here we analyzed whether the transcription of virulence genes that encode T4SS and cellular envelope components, flagellins, adhesins, toxins, urease, as well as the transcription of different regulatory genes of the H. pylori strain 26695, are altered by the presence of five distinct LCFAs: palmitic, stearic, oleic, linoleic, and linolenic acids. Palmitic and oleic acids up-regulated the transcription of most of the virulence genes tested, including cagL, cagM, flaB, sabA, mraY and vacA, as well as that of the genes encoding the transcriptional regulators NikR, Fur, CheY, ArsR, FlgR, HspR, HsrA, Hup, and CrdR. In contrast, the other LCFAs differentially affected the transcription of the virulence and regulatory genes assessed. Our data show that LCFAs can act as signaling molecules that control the transcription of the H. pylori virulome.

An incoherent feedforward loop formed by SirA/BarA, HilE and HilD is involved in controlling the growth cost of virulence factor expression by Salmonella Typhimurium.

An intricate regulatory network controls the expression of Salmonella virulence genes. The transcriptional regulator HilD plays a central role in this network by controlling the expression of tens of genes mainly required for intestinal colonization. Accordingly, the expression/activity of HilD is highly regulated by multiple factors, such as the SirA/BarA two-component system and the Hcp-like protein HilE. SirA/BarA positively regulates translation of hilD mRNA through a regulatory cascade involving the small RNAs CsrB and CsrC, and the RNA-binding protein CsrA, whereas HilE inhibits HilD activity by protein-protein interaction. In this study, we show that SirA/BarA also positively regulates translation of hilE mRNA through the same mentioned regulatory cascade. Thus, our results reveal a paradoxical regulation exerted by SirA/BarA-Csr on HilD, which involves simultaneous opposite effects, direct positive control and indirect negative control through HilE. This kind of regulation is called an incoherent type-1 feedforward loop (I1-FFL), which is a motif present in certain regulatory networks and represents a complex biological problem to decipher. Interestingly, our results, together with those from a previous study, indicate that HilE, the repressor component of the I1-FFL reported here (I1-FFLSirA/BarA-HilE-HilD), is required to reduce the growth cost imposed by the expression of the genes regulated by HilD. Moreover, we and others found that HilE is necessary for successful intestinal colonization by Salmonella. Thus, these findings support that I1-FFLSirA/BarA-HilE-HilD cooperates to control the precise amount and activity of HilD, for an appropriate balance between the growth cost and the virulence benefit generated by the expression of the genes induced by this regulator. I1-FFLSirA/BarA-HilE-HilD represents a complex regulatory I1-FFL that involves multiple regulators acting at distinct levels of gene expression, as well as showing different connections to the rest of the regulatory network governing Salmonella virulence.

Telerehabilitation Programme as a Maintenance Strategy for COPD Patients: A 12-Month Randomized Clinical Trial.

BACKGROUND: There is uncertainty regarding efficacy of telehealth-based approaches in COPD patients for sustaining benefits achieved with intensive pulmonary rehabilitation (PR). RESEARCH QUESTION: To determine whether a maintenance pulmonary telerehabilitation (TelePR) programme, after intensive initial PR, is superior to usual care in sustaining over time benefits achieved by intensive PR. STUDY DESIGN AND METHODS: A multicentre open-label pragmatic parallel-group randomized clinical trial was conducted. Two groups were created at completion of an 8-week intensive outpatient hospital PR programme. Intervention group (IG) patients were given appropriate training equipment and instructed to perform three weekly training sessions and send performance data through an app to a web-based platform. Patients in the control group (CG) were advised to exercise regularly (usual care). RESULTS: Ninety-four patients (46 IG, 48 CG) were randomized. The analysis of covariance showed non-significant improvements in 6-min walk distance [19.9m (95% CI -4.1/+43.8)] and Chronic Respiratory Disease Questionnaire - Emotion score [0.4 points (0-0.8)] in the IG. Secondary linear mixed models showed improvements in the IG in Short Form-36 mental component summary [9.7, (4.0-15.4)] and Chronic Respiratory Disease Questionnaire - Emotion [0.5, (0.2-0.9)] scores, but there was no association between compliance and outcomes. Acute exacerbations were associated with a marginally significant decrease in 6-minute walk distance of 15.8m (-32.3/0.8) in linear models. CONCLUSIONS: The TelePR maintenance strategy was both feasible and safe but failed to show superiority over usual care, despite improvements in some HRQoL domains. Acute exacerbations may have an important negative influence on long-term physical function. CLINICALTRIALS. GOV IDENTIFIER: NCT03247933.

(p)ppGpp-Dependent Regulation of the Nucleotide Hydrolase PpnN Confers Complement Resistance in Salmonella enterica Serovar Typhimurium.

The stringent response is an essential mechanism of metabolic reprogramming during environmental stress that is mediated by the nucleotide alarmones guanosine tetraphosphate and pentaphosphate [(p)ppGpp]. In addition to physiological adaptations, (p)ppGpp also regulates virulence programs in pathogenic bacteria, including Salmonella enterica serovar Typhimurium. S Typhimurium is a common cause of acute gastroenteritis, but it may also spread to systemic tissues, resulting in severe clinical outcomes. During infection, S Typhimurium encounters a broad repertoire of immune defenses that it must evade for successful host infection. Here, we examined the role of the stringent response in S Typhimurium resistance to complement-mediated killing and found that the (p)ppGpp synthetase-hydrolase, SpoT, is required for bacterial survival in human serum. We identified the nucleotide hydrolase, PpnN, as a target of the stringent response that is required to promote bacterial fitness in serum. Using chromatography and mass spectrometry, we show that PpnN hydrolyzes purine and pyrimidine monophosphates to generate free nucleobases and ribose 5'-phosphate, and that this metabolic activity is required for conferring resistance to complement killing. In addition to PpnN, we show that (p)ppGpp is required for the biosynthesis of the very long and long O-antigen in the outer membrane, known to be important for complement resistance. Our results provide new insights into the role of the stringent response in mediating evasion of the innate immune system by pathogenic bacteria.

The Salmonella Typhimurium InvF-SicA complex is necessary for the transcription of sopB in the absence of the repressor H-NS.

Expression of virulence factors in non-typhoidal Salmonella enterica depends on a wide variety of general and specific transcriptional factors that act in response to multiple environmental signals. Expression of genes for cellular invasion located in the Salmonella pathogenicity island 1 (SPI-1) is tightly regulated by several transcriptional regulators arrayed in a cascade, while repression of this system is exerted mainly by H-NS. In SPI-1, H-NS represses the expression mainly by binding to the regulatory region of hilA and derepression is exercised mainly by HilD. However, the possible regulatory role of H-NS in genes downstream from HilD and HilA, such as those regulated by InvF, has not been fully explored. Here the role of H-NS on the expression of sopB, an InvF dependent gene encoded in SPI-5, was evaluated. Our data show that InvF is required for the expression of sopB even in the absence of H-NS. Furthermore, in agreement with previous results on other InvF-regulated genes, we found that the expression of sopB requires the InvF/SicA complex. Our results support that SicA is not required for DNA binding nor for increasing affinity of InvF to DNA in vitro. Moreover, by using a bacterial two-hybrid system we were able to identify interactions between SicA and InvF. Lastly, protein-protein interaction assays suggest that InvF functions as a monomer. Derived from these results we postulate that the InvF/SicA complex does not act on sopB as an anti-H-NS factor; instead, it seems to induce the expression of sopB by acting as a classical transcriptional regulator.

Genomic Analysis Reveals the Genetic Determinants Associated With Antibiotic Resistance in the Zoonotic Pathogen Campylobacter spp. Distributed Globally.

The genus Campylobacter groups 32 Gram-negative bacteria species, several being zoonotic pathogens and a major cause of human gastroenteritis worldwide. Antibiotic resistant Campylobacter is considered by the World Health Organization as a high priority pathogen for research and development of new antibiotics. Genetic elements related to antibiotic resistance in the classical C. coli and C. jejuni species, which infect humans and livestock, have been analyzed in numerous studies, mainly focused on local geographical areas. However, the presence of these resistance determinants in other Campylobacter species, as well as in C. jejuni and C. coli strains distributed globally, remains poorly studied. In this work, we analyzed the occurrence and distribution of antibiotic resistance factors in 237 Campylobacter closed genomes available in NCBI, obtained from isolates collected worldwide, in different dates, from distinct hosts and comprising 22 Campylobacter species. Our data revealed 18 distinct genetic determinants, genes or point mutations in housekeeping genes, associated with resistance to antibiotics from aminoglycosides, ß-lactams, fluoroquinolones, lincosamides, macrolides, phenicols or tetracyclines classes, which are differentially distributed among the Campylobacter species tested, on chromosomes or plasmids. Three resistance determinants, the bla OXA-493 and bla OXA-576 genes, putatively related to ß-lactams resistance, as well as the lnu(AN2) gene, putatively related to lincosamides resistance, had not been reported in Campylobacter; thus, they represent novel determinants for antibiotic resistance in Campylobacter spp., which expands the insight on the Campylobacter resistome. Interestingly, we found that some of the genetic determinants associated with antibiotic resistance are Campylobacter species-specific; e.g., the bla OXA-493 gene and the T86V mutation in gyrA were found only in the C. lari group, whereas genes associated with aminoglycosides resistance were found only in C. jejuni and C. coli. Additional analyses revealed how are distributed the resistance and multidrug resistance Campylobacter genotypes assessed, with respect to hosts, geographical locations, and collection dates. Thus, our findings further expand the knowledge on the factors that can determine or favor the antibiotic resistance in Campylobacter species distributed globally, which can be useful to choose a suitable antibiotic treatment to control the zoonotic infections by these bacteria.

HilD induces expression of a novel Salmonella Typhimurium invasion factor, YobH, through a regulatory cascade involving SprB.

HilD is an AraC-like transcriptional regulator encoded in the Salmonella pathogenicity island 1 (SPI-1), which actives transcription of many genes within and outside SPI-1 that are mainly required for invasion of Salmonella into host cells. HilD controls expression of target genes directly or by acting through distinct regulators; three different regulatory cascades headed by HilD have been described to date. Here, by analyzing the effect of HilD on the yobH gene in Salmonella enterica serovar Typhimurium (S. Typhimurium), we further define an additional regulatory cascade mediated by HilD, which was revealed by previous genome-wide analyses. In this regulatory cascade, HilD acts through SprB, a LuxR-like regulator encoded in SPI-1, to induce expression of virulence genes. Our data show that HilD induces expression of sprB by directly counteracting H-NS-mediated repression on the promoter region upstream of this gene. Then, SprB directly activates expression of several genes including yobH, slrP and ugtL. Interestingly, we found that YobH, a protein of only 79 amino acids, is required for invasion of S. Typhimurium into HeLa cells and mouse macrophages. Thus, our results reveal a novel S. Typhimurium invasion factor and provide more evidence supporting the HilD-SprB regulatory cascade.