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Enterobacter cloacae is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of E. cloacae harbors two T6SS gene clusters (T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in E. cloacae. Single deletions of the genes encoding the HU subunits (hupA and hupB) decreased mRNA levels of both T6SS. In contrast, the hupA hupB double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the hup genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were trans-complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria. IMPORTANCE: T6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche. Enterobacter cloacae expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in E. cloacae, affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on E. cloacae pathogenesis.

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In this work we carried out an in silico analysis to understand the interaction between InvF-SicA and RNAP in the bacterium Salmonella Typhimurium strain LT2. Structural analysis of InvF allowed the identification of three possible potential cavities for interaction with SicA. This interaction could occur with the structural motif known as tetratricopeptide repeat (TPR) 1 and 2 in the two cavities located in the interface of the InvF and α-CTD of RNAP. Indeed, molecular dynamics simulations showed that SicA stabilizes the Helix-turn-Helix DNA-binding motifs, i.e., maintaining their proper conformation, mainly in the DNA Binding Domain (DBD). Finally, to evaluate the role of amino acids that contribute to protein-protein affinity, an alanine scanning mutagenesis approach, indicated that R177 and R181, located in the DBD motif, caused the greatest changes in binding affinity with α-CTD, suggesting a central role in the stabilization of the complex. However, it seems that the N-terminal region also plays a key role in the protein-protein interaction, especially the amino acid R40, since we observed conformational flexibility in this region allowing it to interact with interface residues. We consider that this analysis opens the possibility to validate experimentally the amino acids involved in protein-protein interactions and explore other regulatory complexes where chaperones are involved.

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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.

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We conducted surveillance studies in Sinaloa, Mexico, to determine the circulation of tick-borne relapsing fever spirochetes. We collected argasid ticks from a home in the village of Camayeca and isolated spirochetes. Genomic analysis indicated that Borrelia turicatae infection is a threat to those living in resource-limited settings.

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Soft ticks from the Ornithodoros genus are vectors of relapsing fever (RF) spirochetes around the world. In Mexico, they were originally described in the 19th century. However, few recent surveillance studies have been conducted in Mexico, and regions where RF spirochetes circulate remain vague. Here, the presence of soft ticks in populated areas was assessed in two sites from the Mexican states of Aguascalientes and Zacatecas. Argasidae ticks were collected, identified by morphology and mitochondrial 16S rDNA gene sequencing, and tested for RF borreliae. The specimens in both sites were identified as Ornithodoros turicata but no RF spirochetes were detected. These findings emphasize the need to update the distribution of these ticks in multiple regions of Mexico and to determine the circulation of RF borreliosis in humans and domestic animals.

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Rickettsia species are bacteria that may cause multiple diseases in animals and humans, via transmission through multiple arthropod vectors. Routine surveillance of Rickettsia spp. within vectors is critical to determine their presence and risk to mammalian hosts within human populations. Therefore, to better characterize the circulating Rickettsia species in an understudied region we targeted pet dogs to survey. Ticks were collected from pet dogs in three populations of the Yucatan where we tested for the presence of Rickettsia spp. by PCR in metagenomic DNA. In these ticks removed from pet dogs we detected Rickettsia amblyommatis and Rickettsia bellii in Amblyomma auriculatum, Amblyomma ovale and Amblyomma mixtum ticks obtained in a rural community in the Mexican state of Yucatan. This is the first report detecting both species for this state in Mexico, underpinning the importance of more routine surveillance.

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Relapsing fever (RF) borreliosis is a neglected disease in Mexico. A retrospective serological survey using diagnostic antigens GlpQ and BipA from Borrelia turicatae was performed to evaluate human exposure to RF borreliae. Seventy serum samples were used from a cohort of patients with undifferentiated febrile illness in Mexico. Four samples were positive to GlpQ and three to BipA. Results indicate that RF borreliae continue to circulate in regions of Mexico and pose a risk to human health.

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CRISPR-Cas systems are composed of repeated sequences separated by non-repeated sequences that are near genes coding for Cas proteins, which are involved in the function of these systems. Their function has been mostly related to "genetic immunity" against foreign genetic material, among other roles. Interest in them increased after their use in genetic manipulation was uncovered and surveys to find and classify them have been done in several bacterial groups. To determine the presence of these genetic elements in the Burkholderiaceae family members, a bioinformatic approach was followed. Attention in this family comes as it is formed by a great diversity of microorganisms that include opportunistic and true pathogens, and symbiotic and saprophytic organisms, among others. Results show that, in contrast to other bacterial groups, only 8.4% of family members harbor complete CRISPR-Cas systems and the rest either do not have one or have remains or sections of one. Analyses of the spacer sequences indicated that most of them have identity to sections of the same genomes they were found, while a few had identities with either plasmids or phages. The genus with the higher proportion of self-directed spacers is Ralstonia, and their possible roles are discussed. Most of the systems (60%) belong to the class I subtype I-E and a few to subtypes I-C (13.3%), I-F (18.3%), II-C (5%), IV-A (1.7%) and V-C (1.7%). To the best of our knowledge, this is the first study to uncover the CRISPR-Cas system for the whole Burkholderiaceae family.

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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.

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3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is a crucial enzyme in the ergosterol biosynthesis pathway. The aim of this study was to obtain, purify, characterize, and overexpress five point mutations in highly conserved regions of the catalytic domain of Candida glabrata HMGR (CgHMGR) to explore the function of key amino acid residues in enzymatic activity. Glutamic acid (Glu) was substituted by glutamine in the E680Q mutant (at the dimerization site), Glu by glutamine in E711Q (at the substrate binding site), aspartic acid by alanine in D805A, and methionine by arginine in M807R (the latter two at the cofactor binding site). A double mutation, E680Q-M807R, was included. Regarding recombinant and wild-type CgHMGR, in vitro enzymatic activity was significantly lower for the former, as was the in silico binding energy of simvastatin, alpha-asarone and the HMG-CoA substrate. E711Q displayed the lowest enzymatic activity and binding energy, suggesting the importance of Glu711 (in the substrate binding site). The double mutant CgHMGR E680Q-M807R exhibited the second lowest enzymatic activity. Based on the values of the kinetic parameters KM and Vmax, the mutated amino acids appear to participate in binding. The current findings provide insights into the role of residues in the catalytic site of CgHMGR.

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