Lose to win: marT pseudogenization in Salmonella enterica serovar Typhi contributed to the surV-dependent survival to H2O2, and inside human macrophage-like cells.

The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and Salmonella enterica serovar Typhi (S. Typhi) can be partially attributed to the gain of functions, to the loss of functions (i.e. pseudogenization), or to a combination of both processes. As previously reported, the loss of functions by pseudogenization may play a role in bacterial evolution, especially in host-restricted pathogens such as S. Typhi. The marT-fidL operon, located at the SPI-3, encodes the MarT transcriptional regulator and a hypothetical protein (i.e. FidL) with no significant similarities to known proteins, respectively. Even though predicted S. Typhimurium FidL exhibit 99.4% identity with S. Typhi FidL, marT has been annotated as a pseudogene in S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium marT-fidL exhibited an increased accumulation of reactive oxygen species (ROS), leading to a decreased survival in presence of H2O2. Moreover, we found that that the presence of a functional copy of S. Typhimurium marT-fidL in S. Typhi resulted in a repression of surV (STY4039), an ORF found in the S. Typhi SPI-3 but absent from S. Typhimurium SPI-3, that contribute to the resistance to H2O2 by decreasing the accumulation of ROS. Finally, we observed that the presence of S. Typhimurium marT-fidL in S. Typhi negatively affected the survival inside macrophage-like cells, but not in epithelial cells, after 24h post infection. Therefore, this work provides evidence arguing that marT pseudogenization in Salmonella Typhi contributed to the surV-dependent survival against H2O2, and inside human macrophage-like cells. This is a good example of how the loss of functions (marT pseudogenization) and the gain of functions (presence of surV) might contribute to phenotypic changes improving virulence.

Pseudogenization of sopA and sopE2 is functionally linked and contributes to virulence of Salmonella enterica serovar Typhi.

The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and S. enterica serovar Typhi (S. Typhi) can be partially attributed to pseudogenes. Pseudogenes are genomic segments homologous to functional genes that do not encode functional products due to the presence of genetic defects. S. Typhi lacks several protein effectors implicated in invasion or other important processes necessary for full virulence of S. Typhimurium. SopA and SopE2, effectors that have been lost by pseudogenization in S. Typhi, correspond to an ubiquitin ligase involved in cytokine production by infected cells, and to a guanine exchange factor necessary for invasion of epithelial cells, respectively. We hypothesized that sopA and/or sopE pseudogenization contributed to the virulence of S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium sopE2 exhibited a decreased invasion in different epithelial cell lines compared with S. Typhi WT. S. Typhimurium sopA completely abolished the hypo-invasive phenotype observed in S. Typhi expressing S. Typhimurium sopE2, suggesting that functional SopA and SopE2 participate concertedly in the invasion process. Finally, the expression of S. Typhimurium sopA and/or sopE2 in S. Typhi, determined changes in the secretion of IL-8 and IL-18 in infected epithelial cells.

Salmonella Typhi shdA: pseudogene or allelic variant?

ShdA from Salmonella Typhimurium (ShdASTm) is a large outer membrane protein that specifically recognizes and binds to fibronectin. ShdASTm is involved in the colonization of the cecum and the Peyer's patches of terminal ileum in mice. On the other hand, shdA gene from Salmonella Typhi (shdASTy) has been considered a pseudogene (i.e. a nonfunctional sequence of genomic DNA) due to the presence of deletions and mutations that gave rise to premature stop codons. In this work we show that, despite the deletions and mutations, shdASTy is fully functional. S. Typhi ΔshdA mutants presented an impaired adherence and invasion of HEp-2 pre-treated with TGF-ß1, an inducer of fibronectin production. Moreover, shdA from S. Typhi and S. Typhimurium seem to be equivalent since shdASTm restored the adherence and invasion of S. Typhi ΔshdA mutant to wild type levels. In addition, anti-FLAG mAbs interfered with the adherence and invasion of the S. Typhi shdA-3xFLAG strain. Finally, shdASTy encodes a detectable protein when heterologously expressed in Escherichia coli DH5α. The data presented here show that shdASTy is not a pseudogene, but a different functional allele compared with shdASTm.

Genetic analysis of antibiotic-resistance determinants in multidrug-resistant Shigella strains isolated from Chilean children.

A total of 162 clinical isolates of Shigella collected from children in a semi-rural community of Chile were examined for the presence of genetic determinants of resistance to ampicillin, chloramphenicol, tetracycline, and trimethoprim. Ampicillin resistance was most frequently associated with the presence of bla(OXA) in S. flexneri and with bla(TEM) in S. sonnei. The bla(OXA) gene but not bla(TEM) was located in class 1 integrons. The dhfrIa gene encoding for resistance to trimethoprim was associated to class 2 integrons and detected exclusively in S. flexneri, whereas dhfrIIIc was found in all S. sonnei strains and in 10% of the S. flexneri isolates. Cat, coding for choramphenicol resistance, and bla(OXA) genes were located in the chromosome in all cases, whereas tetA gene, coding for tetracycline resistance, and bla(TEM), dhfrIa and dhfrIIIc genes were found either in the chromosome or in conjugative plasmids. Our results show a heterogenous distribution of antibiotic-resistance determinants between S. flexneri and S. sonnei.

Gene transfer between related bacteria by electrotransformation: mapping Salmonella typhi genes in Salmonella typhimurium.

Transfer of newly isolated mutations into a fresh background is an essential step of genetic analysis and strain construction. Gene transfer is hampered in Salmonella typhi and in other pathogenic bacteria by the lack of a generalized transduction system. We show here that this problem can be partially circumvented by using electrotransformation as a means for delivering S. typhi DNA into suitable S. typhi or Salmonella typhimurium recipients. Transferred DNA can recombine with the homologous region in the host chromosome. In one application of the method, mutations isolated in S. typhi were genetically mapped in S. typhimurium.

Salmonella typhi Ty2 OmpC porin induces bactericidal activity on U937 monocytes.

The immunogenic effect of Salmonella typhi OmpC porin during typhoid fever in humans was evaluated in vitro. Peripheral blood mononuclear cells from 17 patients were challenged with outer membrane preparations from Escherichia coli UH302 and UH302/pSTP2K2 strains, both lacking E. coli OmpF and OmpC porins, although UH302/pSTP2K2 expressed a plasmid-encoded S. typhi Ty2 OmpC. The mononuclear cell supernatants, immunized in vitro with OmpC antigen, derived from 10 out of 17 patients activated U937 bactericidal capacity. In contrast, the supernatants from the immunization with outer membrane preparation lacking S. typhi Ty2 OmpC induced a significantly reduced bactericidal capacity of U937 cells. This procedure should prove useful for in vitro characterization of cellular immunogens from exclusive human pathogens.

Heterologous expression of Escherichia coli porin genes in Salmonella typhi Ty2: regulation by medium osmolarity, temperature and oxygen availability.

Electrophoretic analysis of outer membrane proteins showed that Salmonella typhi OmpC expression is not reciprocally regulated relative to OmpF as described for Escherichia coli and S. typhimurium. When bacteria were grown in minimal media, both OmpC and OmpF were repressed as the osmolarity increased. However, in Luria broth, expression of OmpC was slightly induced by osmolarity up to 0.3 osmM. Plasmids bearing E. coli ompC-lacZ or ompF-lacZ gene fusions were studied for their expression in S. typhi and E. coli. Under anaerobic growth conditions, expression of ompC-lacZ in S. typhi was maximal at 0.16 osmM, while in E. coli expression was maximal at 0.7 osmM. ompF-lacZ expression was similarly repressed by medium osmolarity and anaerobiosis in both species. In contrast, a drastic difference in the regulation of OmpF by temperature was observed; at 37 degrees C ompF-lacZ expression was repressed in E. coli, while in S. typhi it was induced.

Inactivation of mismatch repair overcomes the barrier to transduction between Salmonella typhimurium and Salmonella typhi.

P22 transduction of chromosomal genes from Salmonella typhimurium into Salmonella typhi occurs at a low frequency. Transduction of plasmids from S. typhimurium into S. typhi occurs at a frequency similar to that between S. typhimurium strains, indicating that the barrier to transduction of chromosomal genes is not due to an inability of P22 to inject DNA into S. typhi or a restriction endonuclease that rapidly degrades foreign DNA. Furthermore, transduction of mutS and mutL derivatives of S. typhi with chromosomal genes from S. typhimurium occurs efficiently. These results indicate that the transduction barrier is due to activity of the recipient mismatch repair system, which senses sequence divergence and disrupts heteroduplexes in favor of recipient sequences. Inactivation of the mismatch repair system allows P22 transduction to be used as an effective tool for constructing S. typhi-S. typhimurium hybrids.

Mini-Mu technology in Salmonella typhi: isolation of stable MudJ operon fusions by cis complementation.

This paper describes a method to achieve stable MudJ insertions in the Salmonella typhi Ty2 chromosome. The method is a modification of the genetic complementation system described previously for Salmonella typhimurium, which consists in placing the defective transposon (MudJ) near the transposase genes of a helper Mu phage on a single DNA fragment. This fragment is then introduced into a new bacterial host by means of P22 transduction. We constructed a S. typhi strain which carries MudJ and the Mu helper phage in the chromosome. This strain was induced to lytic growth and the lysate was used to infect S. typhi Ty2. The frequency of mutation was 2.0 x 10(-6) mutants per recipient bacterium. Superinfection with the Mu helper phage was about 1%. To determine the number of MudJ insertions, several mutants were subjected to Southern blot analysis. From a total of 25 mutants analyzed, only 4 contained more than one insertion. Our procedure compares well with the method described previously for the S. typhimurium-P22 system and can be applied to other Mu sensitive bacteria.

Mini-Mu technology in Salmonella typhi: isolation of stable MudJ operon fusions by cis complementation

This paper describes a method to achieve stable MudJ insertions in the Salmonella typhi Ty2 chromosome. The method is a modification of the genetic complementation system described previously for Salmonella typhimurium, which consists in placing the defective transposon (MudJ) near the transposase genes of a helper Mu phage on a single DNA fragment. This fragment is then introduced into a new bacterial host by means of P22 transduction. We constructed a S. typhi strain which carries MudJ and the Mu helper phage in the chromosome. This strain was induced to lytic growth and the lysate was used to infect S. typhi Ty2. The frequency of mutation was 2.0 x 10(-6) mutants per recipient bacterium. Superinfection with the Mu helper phage was about 1 percent. To determine the number of MudJ insertions, several mutants were subjected to Southern blot analysis. From a total of 25 mutants analyzed, only 4 contained more than one insertion. Our procedure compares well with the method described previously for the S. typhimurium-P22 system and can be applied to other Mu sensitive bacteria

Alteration in the electrophoretic mobility of OmpC due to variations in the ammonium persulfate concentration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Studies on Salmonella typhi and Salmonella typhimurium outer membrane proteins have shown that the relative position of OmpC porin in sodium dodecyl sulfate.polyacrylamide gel electrophoresis undergoes an important shift when the concentration of ammonium persulfate in the running gel is increased from 6 to 12 mM. The apparent molecular mass at these concentrations was estimated to be 34 and 40 kDa, respectively. Under similar electrophoretic conditions the apparent molecular mass estimated for OmpF was 37.6 and 38.2 kDa. Therefore, OmpC moves from a leading position to a position behind OmpF. For Escherichia coli OmpC the shift observed is less pronounced than that occurring in Salmonellae.

Clinical isolate of a porinless Salmonella typhi resistant to high levels of chloramphenicol.

We studied a clinical isolate of Salmonella typhi (strain 1895) characterized by resistance to 200 micrograms of chloramphenicol per ml despite the absence of chloramphenicol-inactivating activity. The outer membrane protein profile analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a deficiency of one of the major protein species which may serve as a porin for entry of chloramphenicol. When the strain was transformed with a plasmid encoding chloramphenicol acetyltransferase, chloramphenicol added to the culture was not inactivated, suggesting a drastic reduction of permeability towards the drug. Moreover, transformants bearing a plasmid coding for the Escherichia coli OmpF porin became considerably more susceptible to chloramphenicol (40 micrograms/ml). On the other hand, transformants carrying a plasmid encoding the Salmonella typhi ompC gene remained as resistant to the drug as the parental strain, even though they overexpressed OmpC. These findings indicate that the lack of OmpF plays a major role in the resistance to chloramphenicol in strain 1895.

Antibodies to porin antigens of Salmonella typhi induced during typhoid infection in humans.

Immunoglobulin G (IgG)- and IgM-specific antibody titers against Salmonella typhi Ty2 porins have been measured in 30 paired typhoid sera by enzyme-linked immunosorbent assay. These studies have found that IgG serum titers of acute and convalescent sera were 625 and 5,000 times higher, respectively than the control serum titers. The same typhoid sera were titrated with S. typhi Ty2 flagellin and S. typhi lipopolysaccharide. The titers against these antigens were considerably lower than those against the porins. The highest IgM-specific titer has also been found against porins in convalescent-phase sera. However, the largest increase in IgM-specific titer compared with the control group titer was obtained against flagellin during the acute phase of typhoid. The lowest increases in antibody titer were obtained with the IgM-specific anti-lipopolysaccharide in both types of sera. This may be because many normal individuals in endemic areas already have IgM titers against lipopolysaccharide. This study has provided good evidence that porins are excellent antigens and that IgG-specific antiporin titers may be of diagnostic value in typhoid infections in endemic areas.

The hemolytic effect of Salmonella typhi Ty 2 porins.

Two outer membrane proteins of Salmonella typhi Ty 2 were extensively co-purified. According to their migration in dodecylsulfate/polyacrylamide gel electrophoresis and solubility characteristics, these proteins are homologous to the 35-kDa and 36-kDa porins found in Salmonella typhimurium. A porin homologous to the 34-kDa one has not been found in S. typhi Ty 2. A critical step in the purification of porins is heating at 100 degrees C in 2% sodium dodecyl sulfate before Sephadex gel filtration. The absence of detergent in aqueous suspensions enhances porin aggregation, these aggregations inducing human red cell lysis. Porins obtained by an alternative procedure consisting of heating at 60 degrees C instead of 100 degrees C were also hemolytic. Using nanomolar concentration of porins a strong influence of temperature on the hemolytic effect was observed. Porin-induced hemolysis was inhibited with anti-porin serum, as well as by a treatment with phenylglyoxal, which reacts with the arginine residues of proteins. The membrane-disrupting ability of porins aggregates might explain some pathogenic characteristics of gram-negative bacterial infections.

Immunogenic capacity of ribosomes of Salmonella typhi interfered with a flagellin-like material contaminant.

The double-immunodiffusion technique and sodium dodecyl sulfate-polyacrylamide electrophoresis were used to demonstrate the presence of flagellin-like material strongly attached to ribosomes of Salmonella typhi Ty 2. This flagellin-like material contaminating the ribosome preparation interferes with the induction of antiribosome serum promoting the formation of antisera reacting either only with flagellin or in some cases with flagellin and ribosomes, but giving a very weak reaction with the latter. The interference is also observed when purified ribosomes from a nonflagellated mutant of S. typhi (S. typhi O-901) mixed with purified S. typhi Ty 2 flagellin are utilized as antigens. The antiribosome sera obtained with ribosomes from S. typhi O-901 have a considerably higher titer than those that are interfered with. These sera were able to react with ribosomes obtained from several related species and did not react with flagella-derived flagellin of S. typhi Ty 2.