HilD is an AraC-like transcriptional regulator that plays a central role in Salmonella virulence. HilD controls the expression of the genes within the Salmonella pathogenicity island 1 (SPI-1) and of several genes located outside SPI-1, which are mainly required for Salmonella invasion of host cells. The expression, amount and activity of HilD are tightly controlled by the activities of several factors. The HilE protein represses the expression of the SPI-1 genes through its interaction with HilD; however, the mechanism by which HilE affects HilD is unknown. In this study, we used genetic and biochemical assays revealing how HilE controls the transcriptional activity of HilD. We found that HilD needs to assemble in homodimers to induce expression of its target genes. Our results further indicated that HilE individually interacts with each the central and the C-terminal HilD regions, mediating dimerization and DNA binding, respectively. We also observed that these interactions consistently inhibit HilD dimerization and DNA binding. Interestingly, a computational analysis revealed that HilE shares sequence and structural similarities with Hcp proteins, which act as structural components of type 6 secretion systems in Gram-negative bacteria. In conclusion, our results uncover the molecular mechanism by which the Hcp-like protein HilE controls dimerization and DNA binding of the virulence-promoting transcriptional regulator HilD. Our findings may indicate that HilE's activity represents a functional adaptation during the evolution of Salmonella pathogenicity.

A wide variety of Salmonella enterica serovars cause intestinal and systemic infections to humans and animals. Salmonella Patogenicity Island 1 (SPI-1) is a chromosomal region containing 39 genes that have crucial virulence roles. The AraC-like transcriptional regulator HilD, encoded in SPI-1, positively controls the expression of the SPI-1 genes, as well as of several other virulence genes located outside SPI-1. In this study, we applied a clustering method to the global gene expression data of S. enterica serovar Typhimurium from the COLOMBOS database; thus genes that show an expression pattern similar to that of SPI-1 genes were selected. This analysis revealed nine novel genes that are co-expressed with SPI-1, which are located in different chromosomal regions. Expression analyses and protein-DNA interaction assays showed regulation by HilD for six of these genes: gtgE, phoH, sinR, SL1263 (lpxR) and SL4247 were regulated directly, whereas SL1896 was regulated indirectly. Interestingly, phoH is an ancestral gene conserved in most of bacteria, whereas the other genes show characteristics of genes acquired by Salmonella. A role in virulence has been previously demonstrated for gtgE, lpxR and sinR. Our results further expand the regulon of HilD and thus identify novel possible Salmonella virulence genes.

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Beyond the development of resistance, the effects of antibiotics on bacteria and microbial communities are complex and far from exhaustively studied. In the context of the current global antimicrobial resistance crisis, understanding the adaptive and physiological responses of bacteria to antimicrobials is of paramount importance along with the development of new therapies. Bacterial dependence on antibiotics is a phenomenon in which antimicrobials instead of eliminating the pathogens actually provide a boost for their growth. This trait comprises an extreme example of the complexities of responses elicited by microorganisms to these drugs. This compelling evolutionary trait was readily described along with the first wave of antibiotics use and dependence to various antimicrobials has been reported. Nevertheless, current molecular characterizations have been focused on dependence on vancomycin, linezolid and colistin, three critically important antibiotics frequently used as last resource therapy for multi resistant pathogens. Outstanding advances have been made in understanding the molecular basis for the dependence to vancomycin, including specific mutations involved. Regarding linezolid and colistin, the general physiological components affected by the dependence, namely ribosomes and membrane function respectively, have been established. Nonetheless the implications of antibiotic dependence in clinically relevant features, such as virulence, epidemics, relationship with development of resistance, diagnostics and therapy effectiveness require clarification. This review presents a brief introduction of the phenomenon of bacterial dependence to antibiotics and a summary on early and current research concerning the basis for this trait. Furthermore, the available information on the effect of dependence in key clinical aspects is discussed. The studies performed so far underline the need to fully disclose the biological and clinical significance of this trait in pathogens to successfully assess its role in resistance and to design adjusted therapies.

Objetivo: Implementar una técnica de PCR (Reacción en cadena de la polimerasa) para la detección de Chlamydia trachomatis. Materiales y Métodos: Se realizó un estudio experimental en el que se estandarizaron las condiciones de PCR (Concentración de MgCl2 , Taq polimerasa y temperatura de alineamiento de cebadores) para la amplificación de una región de 201pb del plásmido de C. trachomatis con los cebadores CtP1 5´-TAGTAACTGCCACTTCATCA-3´ y CtP2 5´- TTCCCCTTGTAATTCGTTGC-3. Se realizaron diluciones seriadas del ADN de C. trachomatis ATCC VR885D para determinar la sensibilidad analítica. La especificidad analítica de los cebadores se determinó con ADN de diferentes microorganismos patógenos y comensales del tracto urogenital. Se determinó la variabilidad intra e interensayo de la PCR sobre triplicados de diferentes muestras de ADN. Resultados: Las condiciones de amplificación fueron 94°C/4 min, seguido de 40 ciclos a 94°C/1 min, 56°C/1 min y 72°C/1.5 min y extensión final de 72°C/4 min. La concentración óptima de MgCl2 fue 1.5 mM y de ADN polimerasa 1U. La sensibilidad analítica de la prueba fue 4.8 x10-15g/mL de ADN equivalentes a 160 cuerpos elementales de C. trachomatis. La especificidad analítica fue 100% y la variabilidad intra e interensayo mostraron reproducibilidad de la PCR. Conclusiones: Los datos sugieren que esta PCR puede emplearse con fines diagnósticos. Se requieren estudios adicionales para la evaluación clínica de esta prueba.

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