Evolutionary History and Strength of Selection Determine the Rate of Antibiotic Resistance Adaptation.

Bacterial adaptation to stressful environments often produces evolutionary constraints whereby increases in resistance are associated with reduced fitness in a different environment. The exploitation of this resistance-cost trade-off has been proposed as the basis of rational antimicrobial treatment strategies designed to limit the evolution of drug resistance in bacterial pathogens. Recent theoretical, laboratory, and clinical studies have shown that fluctuating selection can maintain drug efficacy and even restore drug susceptibility, but can also increase the rate of adaptation and promote cross-resistance to other antibiotics. In this paper, we combine mathematical modeling, experimental evolution, and whole-genome sequencing to follow evolutionary trajectories towards ß-lactam resistance under fluctuating selective conditions. Our experimental model system consists of eight populations of Escherichia coli K12 evolving in parallel to a serial dilution protocol designed to dynamically control the strength of selection for resistance. We implemented adaptive ramps with mild and strong selection, resulting in evolved populations with similar levels of resistance, but with different evolutionary dynamics and diverging genotypic profiles. We found that mutations that emerged under strong selection are unstable in the absence of selection, in contrast to resistance mutations previously selected in the mild selection regime that were stably maintained in drug-free environments and positively selected for when antibiotics were reintroduced. Altogether, our population dynamics model and the phenotypic and genomic analysis of the evolved populations show that the rate of resistance adaptation is contingent upon the strength of selection, but also on evolutionary constraints imposed by prior drug exposures.

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.

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.

(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.

SlyA and HilD Counteract H-NS-Mediated Repression on the ssrAB Virulence Operon of Salmonella enterica Serovar Typhimurium and Thus Promote Its Activation by OmpR.

H-NS-mediated repression of acquired genes and the subsequent adaptation of regulatory mechanisms that counteract this repression have played a central role in the Salmonella pathogenicity evolution. The Salmonella pathogenicity island 2 (SPI-2) is an acquired chromosomal region containing genes necessary for Salmonella enterica to colonize and replicate in different niches of hosts. The ssrAB operon, located in SPI-2, encodes the two-component system SsrA-SsrB, which positively controls the expression of the SPI-2 genes but also other many genes located outside SPI-2. Several regulators have been involved in the expression of ssrAB, such as the ancestral regulators SlyA and OmpR, and the acquired regulator HilD. In this study, we show how SlyA, HilD, and OmpR coordinate to induce the expression of ssrAB under different growth conditions. We found that when Salmonella enterica serovar Typhimurium is grown in nutrient-rich lysogeny broth (LB), SlyA and HilD additively counteract H-NS-mediated repression on ssrAB, whereas in N-minimal medium (N-MM), SlyA antagonizes H-NS-mediated repression on ssrAB independently of HilD. Interestingly, our results indicate that OmpR is required for the expression of ssrAB independently of the growth conditions, even in the absence of repression by H-NS. Therefore, our data support two mechanisms adapted for the expression of ssrAB under different growth conditions. One involves the additive action of SlyA and HilD, whereas the other involves SlyA, but not HilD, to counteract H-NS-mediated repression on ssrAB, thus favoring in both cases the activation of ssrAB by OmpR.IMPORTANCE The global regulator H-NS represses the expression of acquired genes and thus avoids possible detrimental effects on bacterial fitness. Regulatory mechanisms are adapted to induce expression of the acquired genes in particular niches to obtain a benefit from the information encoded in the foreign DNA, as for pathogenesis. Here, we show two mechanisms that were integrated for the expression of virulence genes in Salmonella Typhimurium. One involves the additive action of the regulators SlyA and HilD, whereas the other involves SlyA, but not HilD, to counteract H-NS-mediated repression on the ssrAB operon, thus favoring its activation by the OmpR regulator. To our knowledge, this is the first report involving the coordinated action of two regulators to counteract H-NS-mediated repression.

The transcriptional regulator SsrB is involved in a molecular switch controlling virulence lifestyles of Salmonella.

The evolution of bacterial pathogenicity, heavily influenced by horizontal gene transfer, provides new virulence factors and regulatory connections that alter bacterial phenotypes. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) are chromosomal regions that were acquired at different evolutionary times and are essential for Salmonella virulence. In the intestine of mammalian hosts, Salmonella expresses the SPI-1 genes that mediate its invasion to the gut epithelium. Once inside the cells, Salmonella down-regulates the SPI-1 genes and induces the expression of the SPI-2 genes, which favor its intracellular replication. The mechanism by which the invasion machinery is deactivated following successful invasion of host cells is not known. Here, we show that the SPI-2 encoded transcriptional regulator SsrB, which positively controls SPI-2, acts as a dual regulator that represses expression of SPI-1 during intracellular stages of infection. The mechanism of this SPI-1 repression by SsrB was direct and acts upon the hilD and hilA regulatory genes. The phenotypic effect of this molecular switch activity was a significant reduction in invasion ability of S. enterica serovar Typhimurium while promoting the expression of genes required for intracellular survival. During mouse infections, Salmonella mutants lacking SsrB had high levels of hilA (SPI-1) transcriptional activity whereas introducing a constitutively active SsrB led to significant hilA repression. Thus, our results reveal a novel SsrB-mediated mechanism of transcriptional crosstalk between SPI-1 and SPI-2 that helps Salmonella transition to the intracellular lifestyle.

The global regulatory system Csr senses glucose through the phosphoenolpyruvate: carbohydrate phosphotransferase system.

A novel connection between two regulatory systems controlling crucial biological processes in bacteria, the carbon storage regulator (Csr) system and the glucose-specific phosphotransferase system (PTS), is reported by Leng et al. in this issue. This involves the interaction of unphosphorylated EIIA(Glc), a component of the glucose-specific PTS, with the CsrD protein, which accelerates the decay of the CsrB and CsrC small RNAs via RNase E in Escherichia coli. As unphosphorylated EIIA(G) (lc) is generated in the presence of glucose, the PTS thus acts as a sensor of glucose for the Csr system. Interestingly, another pathway can operate for communication between the Csr system and the glucose-specific PTS. The absence of glucose generates phosphorylated EIIA(Glc) , which activates the enzyme adenylate cyclase to produce cyclic adenosine monophosphate (cAMP) that, in turn, binds to the regulator cAMP receptor protein (CRP). Leng et al. show that the complex cAMP-CRP modestly reduces CsrB decay independently of CsrD. On the other hand, a previous study indicates that the complex cAMP-CRP positively regulates the transcription of CsrB and CsrC in Salmonella enterica. Therefore, EIIA(G) (lc) could work as a molecular switch that regulates the activity of the Csr system, in response to its phosphorylation state determined by the presence or absence of glucose, in order to control gene expression.

A multi-drug resistant Salmonella Typhimurium ST213 human-invasive strain (33676) containing the bla CMY-2 gene on an IncF plasmid is attenuated for virulence in BALB/c mice.

BACKGROUND: Classical strains of Salmonella enterica serovar Typhimurium (Typhimurium) predominantly cause a self-limiting diarrheal illness in humans and a systemic disease in mice. In this study, we report the characterization of a strain isolated from a blood-culture taken from a 15-year old woman suffering from invasive severe salmonellosis, refractory to conventional therapy with extended-spectrum cephalosporin (ESC). RESULTS: The strain, named 33676, was characterized as multidrug-resistant Salmonella serogroup A by biochemical, antimicrobial and serological tests. Multilocus sequence typing (MLST) and XbaI macrorestrictions (PFGE) showed that strain 33676 belonged to the Typhimurium ST213 genotype, previously described for other Mexican Typhimurium strains. PCR analyses revealed the presence of IncA/C, IncFIIA and ColE1-like plasmids and the absence of the Salmonella virulence plasmid (pSTV). Conjugation assays showed that the ESC-resistance gene bla CMY-2 was carried on the conjugative IncF plasmid, instead of the IncA/C plasmid, as found in previously studied ST213 strains. Although the IncA/C plasmid conferred most of the observed antimicrobial resistances it was not self-conjugative; it was rather able to conjugate by co-integrating with the IncF plasmid. Strain 33676 was fully attenuated for virulence in BALB/c mice infections. Both type-three secretion system (T3SS), encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), were functional in the 33676 strain and, interestingly, this strain produced the H2 FljB flagellin instead of the H1 FliC flagellin commonly expressed by S. enterica strains. CONCLUSIONS: Strain 33676 showed two main features that differentiate it from the originally described ST213 strains: 1) the bla CMY-2 gene was not carried on the IncA/C plasmid, but on a conjugative IncF plasmid, which may open a new route of dissemination for this ESC-resistance gene, and 2) it expresses the H2 FljB flagella, in contrast with the other ST213 and most Typhimurium reference strains. To our knowledge this is the first report of an IncF bla CMY-2-carrying plasmid in Salmonella.

Seroprevalence and major antigens recognized by sera from Trypanosoma cruzi-infected dogs from Jalisco, México.

Chagas disease is a major endemic disease caused by the protozoan parasite Trypanosoma cruzi. This parasitic disease is widely distributed throughout Latin America, affecting 10 million people. There are also reports of canine infection in the southern part of the United States. Dogs are considered the predominant domestic reservoir for T. cruzi in many areas of endemicity. In México, dog infection by this parasite has been poorly studied. In this work 209 dogs from six villages in Jalisco, México, were assessed to detect anti-T. cruzi antibodies by ELISA and Western blot. Seventeen (17) seropositive dogs (8.1 %) were detected by both tests, representing a seropositive value similar to that found in some southern states of México where the infection is present. No statistical differences were observed concerning the age and sex of infected and non-infected dogs. The major antigens recognized by positive sera were 26, 32, 66 and 80kDa. These proteins are candidates to develop a specific diagnostic method for canine Chagas. No antibodies against HSP16 protein were found in T. cruzi seropositive sera. This is the first report of canine serology of Chagas disease in this central part of México. This report will contribute to the knowledge of the infection status of domestic reservoirs in the state of Jalisco, México.

Proteomic analysis of Trypanosoma cruzi epimastigotes subjected to heat shock.

Trypanosoma cruzi is exposed to sudden temperature changes during its life cycle. Adaptation to these variations is crucial for parasite survival, reproduction, and transmission. Some of these conditions may change the pattern of genetic expression of proteins involved in homeostasis in the course of stress treatment. In the present study, the proteome of T. cruzi epimastigotes subjected to heat shock and epimastigotes grow normally was compared by two-dimensional gel electrophoresis followed by mass spectrometry for protein identification. Twenty-four spots differing in abundance were identified. Of the twenty-four changed spots, nineteen showed a greater intensity and five a lower intensity relative to the control. Several functional categories of the identified proteins were determined: metabolism, cell defense, hypothetical proteins, protein fate, protein synthesis, cellular transport, and cell cycle. Proteins involved in the interaction with the cellular environment were also identified, and the implications of these changes are discussed.

Changes in cyst's nuclear chromatin resulting after experimental manipulation of Taenia crassiceps mice infections: biological implications.

During some estimations of the nuclear DNA content, based on determinations propidium iodide (PI) binding through fluorocytometry for Taenia crassiceps cysticerci, significant variation in the results were found. This initial observation led to a series of experiments designed to explain the variation. These changes could be induced by the diameter of the needles in the syringes used for the mouse to mouse transfer of the cysts. Nuclei from cysts transferred through 27-gauge needles showed 30% less PI staining than those transferred through 21 gauge needles, after 2 months infections. Reduction in PI capture induced by 27-gauge needle was reversible when the cysts were maintained in their mice hosts during 5 months. Moreover, variation in PI binding to cysticercal DNA was also found when comparing parasites grown in male versus female mice. The use of agents that homogenize the chromatin structure during PI staining, allowed demonstrating that variation were entirely due to differences in the chromatin relaxation/compaction. Additional experiments demonstrated that the higher compaction is accompanied by a reduced ability of cysts to grow in the peritoneal cavity of BALB/cAnN mice. Furthermore, proteomic analysis also showed that these changes in chromatin relaxation/compaction resulted in different levels and patterns of protein expression. Our results strongly suggest that chromatin is involved in several well characterized phenomena of the T. crassiceps murine model, and open new avenues for a detailed approach to understand such a complex host-parasite relationship.

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.

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.

Trypanosoma cruzi SHSP16: Characterization of an alpha-crystallin small heat shock protein.

This report describes the characterization of a member of the alpha-crystallin small heat shock protein family in a trypanosomatid, which was isolated from the human pathogen Trypanosoma cruzi. One alpha-crystallin small heat shock protein gene was identified in a database search. The coding region is located in an open reading frame of 429bp encoding a protein of 142 amino acids. The amino acid sequence was deduced from the isolated gene. The protein has an alpha-crystallin domain characteristic of the alpha-crystallin small heat shock proteins and a molecular weight of 15.9kDa, so the protein was designated SHSP16. Analysis of the nucleotide sequences of four different T. cruzi strains showed two different sequences, which correspond to the two main T. cruzi genetic groups. Gene expression analysis by RT-PCR showed increased transcription of the gene after the parasite was exposed to heat stress. Recombinant SHSP16 showed molecular chaperone activity in vitro, because it inhibited the thermal aggregation of the mitochondrial malate dehydrogenase enzyme.

Regulatory Evolution Drives Evasion of Host Inflammasomes by Salmonella Typhimurium.

Bacterial two-component regulatory systems (TCS) couple the detection of niche-specific cues with adaptive gene expression to optimize fitness. In Salmonella Typhimurium (STM), the SsrA-SsrB TCS regulates virulence genes needed for survival within host cells, yet the impact of this TCS on regulatory evolution in this pathogen remains incompletely understood. Here, we show that SsrB alters a transcriptional network controlling bacterial motility to limit inflammasome activation during host cell infection. Using comparative RNA sequencing between STM and S. bongori (SBG) engineered to express SsrB, we show that SsrB represses flagellar gene expression in STM but activates this pathway in SBG, which has evolved in the absence of SsrB. Motility repression in STM is driven by an SsrB-binding region upstream of flhDC that appears to have evolved in STM following divergence from SBG. These data reveal a divergent regulatory circuit in non-coding DNA that reduces flagellar gene expression to evade host defenses.

Ultrastructural and physiological changes induced by different stress conditions on the human parasite Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease. The life cycle of this protozoan parasite is digenetic because it alternates its different developmental forms through two hosts, a vector insect and a vertebrate host. As a result, the parasites are exposed to sudden and drastic environmental changes causing cellular stress. The stress response to some types of stress has been studied in T. cruzi, mainly at the molecular level; however, data about ultrastructure and physiological state of the cells in stress conditions are scarce or null. In this work, we analyzed the morphological, ultrastructural, and physiological changes produced on T. cruzi epimastigotes when they were exposed to acid, nutritional, heat, and oxidative stress. Clear morphological changes were observed, but the physiological conditions varied depending on the type of stress. The maintenance of the physiological state was severely affected by heat shock, acidic, nutritional, and oxidative stress. According to the surprising observed growth recovery after damage by stress alterations, different adaptations from the parasite to these harsh conditions were suggested. Particular cellular death pathways are discussed.

In silico clustering of Salmonella global gene expression data reveals novel genes co-regulated with the SPI-1 virulence genes through HilD.

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.

The role of small heat shock proteins in parasites.

The natural life cycle of many protozoan and helminth parasites involves exposure to several hostile environmental conditions. Under these circumstances, the parasites arouse a cellular stress response that involves the expression of heat shock proteins (HSPs). Small HSPs (sHSPs) constitute one of the main families of HSPs. The sHSPs are very divergent at the sequence level, but their secondary and tertiary structures are conserved and some of its members are related to α-crystallin from vertebrates. They are involved in a variety of cellular processes. As other HSPs, the sHSPs act as molecular chaperones; however, they have shown other activities apparently not related to chaperone action. In this review, the diverse activities of sHSPs in the major genera of protozoan and helminth parasites are described. These include stress response, development, and immune response, among others. In addition, an analysis comparing the sequences of sHSPs from some parasites using a distance analysis is presented. Because many parasites face hostile conditions through its life cycles the study of HSPs, including sHSPs, is fundamental.