The two-component sensor kinase KdpD is required for Salmonella typhimurium colonization of Caenorhabditis elegans and survival in macrophages

The ability of enteric pathogens to perceive and adapt to distinct environments within the metazoan intestinal tract is critical for pathogenesis; however, the preponderance of interactions between microbe- and host-derived factors remain to be fully understood. Salmonella enterica serovar Typhimurium is a medically important enteric bacterium that colonizes, proliferates and persists in the intestinal lumen of the nematode Caenorhabditis elegans. Several Salmonella virulence factors important in murine and tissue culture models also contribute to worm mortality and intestinal persistence. For example, PhoP and the virulence plasmid pSLT are virulence factors required for resistance to the C. elegans antimicrobial peptide SPP-1. To uncover additional determinants required for Salmonella typhimurium pathogenesis in vivo, we devised a genetic screen to identify bacterial mutants defective in establishing a persistent infection in the intestine of C. elegans. Here we report on identification of 14 loci required for persistence in the C. elegans intestine and characterization of KdpD, a sensor kinase of a two-component system in S. typhimurium pathogenesis. We show that kdpD mutants are profoundly attenuated in intestinal persistence in the nematode and in macrophage survival. These findings may be attributed to the essential role KdpD plays in promoting resistance to osmotic, oxidative and antimicrobial stresses.     

Resistance to antimicrobial peptides contributes to persistence of Salmonella typhimurium in the C. elegans intestine

The human pathogen Salmonella typhimurium can colonize, proliferate and persist in the intestine causing enteritis in mammals and mortality in the nematode Caenorhabditis elegans. Using C. elegans as a model, we determined that the Salmonella pathogenicity islands-1 and -2 (SPI-1 and SPI-2), PhoP and the virulence plasmid are required for the establishment of a persistent infection. We observed that the PhoP regulon, SPI-1, SPI-2 and spvR are induced in C. elegans and isogenic strains lacking these virulence factors exhibited significant defects in the ability to persist in the worm intestine. Salmonella infection also leads to induction of two C. elegans antimicrobial genes, abf-2 and spp-1, which act to limit bacterial proliferation. The SPI-2, phoP and Delta pSLT mutants are more sensitive to the cationic peptide polymyxin B, suggesting that resistance to worm's antimicrobial peptides might be necessary for Salmonella to persist in the C. elegans intestine. Importantly, we showed that the persistence defects of the SPI-2, phoP and Delta pSLT mutants could be rescued in vivo when expression of C. elegans spp-1 was reduced by RNAi. Together, our data suggest that resistance to host antimicrobials in the intestinal lumen is a key mechanism for Salmonella persistence.     

Demonstration of cel operon expression of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa at elevated temperatures refractory to their growth.

When Escherichia coli was incubated at the growth-refractory temperatures of 48 and 54 degrees C, expression of the cel operon was demonstrated by phospho-beta-glucosidase activity. This enzyme activity was also detected at the growth-refractory temperatures in Salmonella typhimurium and Pseudomonas aeruginosa. Thermotolerant and mesothermophilic mutants of E. coli, S. typhimurium, and P. aeruginosa, able to grow with generation times of 30 to 40 min at 48 and 54 degrees C, exhibited phospho-beta-glucosidase activity at their growth temperatures of 48 and 54 degrees C. Thus, the cel operon previously described as a cryptic operon in E. coli and S. typhimurium was found to be expressed at growth-refractory temperatures of the mesophilic parent and growth-permissive temperatures (48 and 54 degrees C) of the thermotolerant and mesothermophilic mutants.     

Demonstration of cel operon expression of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa at elevated temperatures refractory to their growth.

When Escherichia coli was incubated at the growth-refractory temperatures of 48 and 54 degrees C, expression of the cel operon was demonstrated by phospho-beta-glucosidase activity. This enzyme activity was also detected at the growth-refractory temperatures in Salmonella typhimurium and Pseudomonas aeruginosa. Thermotolerant and mesothermophilic mutants of E. coli, S. typhimurium, and P. aeruginosa, able to grow with generation times of 30 to 40 min at 48 and 54 degrees C, exhibited phospho-beta-glucosidase activity at their growth temperatures of 48 and 54 degrees C. Thus, the cel operon previously described as a cryptic operon in E. coli and S. typhimurium was found to be expressed at growth-refractory temperatures of the mesophilic parent and growth-permissive temperatures (48 and 54 degrees C) of the thermotolerant and mesothermophilic mutants.     

The nematode Panagrellus redivivus is susceptible to killing by human pathogens at 37 degrees C

Caenorhabditis elegans has been used as a host for the study of bacteria that cause disease in mammals. However, a significant limitation of the model is that C. elegans is not viable at 37 degrees C. We report that the gonochoristic nematode Panagrellus redivivus survives at 37 degrees C and maintains its life cycle at temperatures up to and including 31.5 degrees C. The C. elegans pathogens Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, but not Yersinia pseudotuberculosis, reduced P. redivivus lifespan. Of four strains of Burkholderia multivorans tested, one reduced P. redivivus lifespan at both temperatures, one was avirulent at both temperatures and two strains reduced P. redivivus lifespan only at 37 degrees C. The mechanism by which one of these strains killed P. redivivus at 37 degrees C, but not at 25 degrees C, was investigated further. Killing required viable bacteria, did not involve bacterial invasion of tissues, is unlikely to be due to a diffusible, bacterial toxin and was not associated with increased numbers of live bacteria within the intestine of the worm. We believe B. multivorans may kill P. redivivus by a temperature-regulated mechanism similar to B. pseudomallei killing of C. elegans.     

One-step cloning system for isolation of bacterial lexA-like genes.

A system to isolate lexA-like genes of bacteria directly was developed. It is based upon the fact that the presence of a lexA(Def) mutation is lethal to SulA+ cells of Escherichia coli. This system is composed of a SulA- LexA(Def) HsdR- strain and a lexA-conditional killer vector (plasmid pUA165) carrying the wild-type sulA gene of E. coli and a polylinker in which foreign DNA may be inserted. By using this method, the lexA-like genes of Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa, and P. putida were cloned. We also found that the LexA repressor of S. typhimurium presented the highest affinity for the SOS boxes of E. coli in vivo, whereas the LexA protein of P. aeruginosa had the lowest. Likewise, all of these LexA repressors were cleaved by the activated RecA protein of E. coli after DNA damage. Furthermore, under high-stringency conditions, the lexA gene of E. coli hybridized with the lexA genes of S. typhimurium and E. carotovora but not with those of P. aeruginosa and P. putida.     

Differential interactions within the Caenorhabditis elegans-Pseudomonas aeruginosa pathogenesis model

A pathogenesis model based on the interaction between Caenorhabditis elegans and bacterial opportunistic pathogens has recently been developed. In the case of Pseudomonas aeruginosa, the model is based on three different modes of nematode killing (fast killing, slow killing and lethal paralysis) by virulent bacteria that has been incubated in different nutrient media. Using parametric statistics and Probit analysis, we test the reliability of the three different killing systems with respect to bacterial virulence. To accomplish this, we use three P. aeruginosa strains, each with a different level of virulence and one strain of non-virulent Escherichia coli. Probit function proved to be effective in quantifying the virulence of P. aeruginosa. The results of the killing curve analysis using the Probit function demonstrates that the slow-killing test is the most reliable method for quantifying virulence using the C. elegans model of bacterial pathogenesis. Although the greatest virulence differences are observed after long periods of incubation, the Probit analysis clearly shows that the death kinetics of C. elegans depend on the first hours of nematode/bacteria interaction. In contrast, fast killing seems to be non-specific, at least under our experimental conditions, since the killing rates of virulent P. aeruginosa and non-virulent E. coli strains were indistinguishable.     

In vitro inhibitory effect of cranberry (Vaccinium macrocarpom Ait.) juice on pathogenic microorganisms

The purpose of this study was to determine the inhibitory effects of cranberry juice on pathogenic microorganisms. The microorganisms analyzed were Escherichia coli from patients with urinary infections, Salmonella spp., Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. The disc method was used to determine the sensitivity of bacteria to cranberry juice (CJ, both concentrated and diluted). A lawn of 10(6) cfu/ml was grown on agar surfaces in Petri dishes and on Whatman discs that had been previously saturated with CJ and CJ : water 1 : 1 to 1 : 50 juice solutions had been placed on the discs, which were cultured and incubated. The results indicated that S. aureus was more susceptible to cranberry juice inhibition than the other microorganisms. L. monocytogenes was the most resistant to the inhibitory action of cranberry juice, showing a significant difference from the inhibition of P. aeruginosa, uropathogenic E. coli, Salmonella spp., and S. aureus. This study also demonstrated that the inhibitory activity of cranberry juice for E. coli took place up to a dilution of 1 : 20.     

Genes affecting coliphage BF23 and E colicin sensitivity in Salmonella typhimurium.

Rough strains of Salmonella typhimurium were sensitive to coliphage BF23. Spontaneous mutants resistant to BF23 (bfe) were isolated, and the trait was mapped using phage P1. The bfe gene in S. typhimurium was located between argF (66% co-transducible) and rif (61% co-transducible). The BF23-sensitive S. typhimurium strains were not sensitive to the E colicins. Cells of these rough strains absorbed colicin, as measured by loss of E2 or E3 killing units from colicin solutions and by specific adsorption of 125I-colicin E2 to bfe+ cells. Sensitivity to colicins E1, E2, and E3 was observed in a S. typhimurium strain carrying the F'8 gal+ episome. This episome complemented the tolB mutation of Escherichia coli. We conclude that the bfe+ protein satisfies requirements for adsorption of both phage BF23 and the E colicins. In addition, expression of a gene from E. coli, possibly tolB, is necessary for efficient E colicin killing of S. typhimurium.     

Genetic analysis of insertion mutations of the promiscuous IncP-1 plasmid R18 mapping near oriT which affect its host range

Transposon Tn7 insertion mutations of the promiscuous IncP-1 plasmid R18 which affect its conjugational transmissibility from Pseudomonas aeruginosa to Escherichia coli C, a strain of E. coli K12, Salmonella typhimurium and P. maltophilia have been mapped physically. They map to coordinate 53.5 kb in the Tral region of the plasmid. An 800-bp fragment mapping between R18 coordinates 52.85 and 53.65 kb, which complemented the host range defect of the mutants when tested with E. coli C as recipient, has been identified. However, complementation occurred only when the 800-bp cloned fragment was provided in the E. coli C recipient but not when situated in the P. aeruginosa donor. It is concluded that a trans-acting gene product of R18 is required, in the transcipient, for conjugative DNA metabolism during, or immediately following, the conjugational transfer of this plasmid between certain donor and recipient hosts.     

Existence of a Free Form of a Specific Membrane Lipoprotein in Gram-Negative Bacteria

The existence of a free form of a specific lipoprotein of molecular weight 7,200 was examined in the envelopes of several gram-negative bacteria. When the envelope proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, distinct peaks were observed in Salmonella typhimurium, Serratia marcescens, and Pseudomonas aeruginosa at the same position as the free form of the lipoprotein of Escherichia coli. However, the peak was not observed in Proteus mirabilis. The protein at the peak in S. typhimurium was shown to contain little or no histidine as expected from the amino acid composition of the lipoprotein. Furthermore, antiserum against the highly purified lipoprotein from E. coli was shown to react with the proteins from S. typhimurium and S. marcescens and to form the specific immunoprecipitates. In contrast, the protein from P. aeruginosa did not react with the antiserum at all. Thus, it is concluded that S. typhimurium and S. marcescens have the free form of the lipoprotein in their envelopes as does E. coli. P. aeruginosa contains a protein of the same size as the lipoprotein, but it is not certain whether the protein is the same structural protein as the lipoprotein from E. coli. P. mirabilis may not have any free form of the lipoprotein, may have it in a very small amount, or may have a lipoprotein of different molecular weight serving the same function.     

A minimal mechanism for bacterial pattern formation

Colonies of Escherichia coli or Salmonella typhimurium form geometrically complex patterns when exposed to, or feeding on, intermediates of the tricarboxylic acid (TCA) cycle. In response to the TCA cycle intermediate, the bacteria secrete aspartate, a potent chemo-attractant. As a result, the cells form high-density aggregates arranged in striking regular patterns. The simplest are temporary spots formed in a liquid medium by both E. coli and S. typhimurium. In semi-solid medium S. typhimurium forms concentric rings arising from a low-density bacterial lawn, which are either continuous or spotted, whereas E. coli forms complex patterns arising from a dense swarm ring, including interdigitated spots (also called sunflower spirals), radial spots, radial stripes and chevrons. We present a mathematical model that captures all three of the pattern-forming processes experimentally observed in both E. coli and S. typhimurium, using a minimum of assumptions.     

Antibacterial activity of the metabolic by-products of a Veillonella species and Bacteroides fragilis

A Veillonella species and Bacteroides fragilis were isolated from the cecal contents of adult chickens. When growth on an agar medium supplemented with 0.4% glucose and adjusted to pH 6.5, mixed cultures containing Veillonella and B. fragilis inhibited the growth of Salmonella typhimurium; Salmonella enteritidis, Escherichia coli 0157:H7 and Pseudomonas aeruginosa. Decreasing the glucose concentration of the agar decreased the inhibitory activity of the mixed culture. Mixed cultures grown on agar media supplemented with 0.5% glucose and adjusted to pH 6.5, 7.0 or 7.5 also inhibited the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 and P. aeruginosa. However, increasing the pH of the agar decreased the inhibitory activity of the mixed culture. Pure cultures of Veillonella or B. fragilis did not inhibit the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 or P. aeruginosa on any of the agar supplemented with different concentrations of glucose or on any of the agar adjusted to different pH levels. The inhibitory activity of the mixed culture was correlated with the concentration of volatile fatty acids that were formed as B. fragilis metabolized glucose to produce succinate and acetate and as the succinate produced by B. fragilis was decarboxylated by Veillonella to produce propionate.     

An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp

The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonella typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-1 and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus.     

Growth of Coliphage T7 in Salmonella typhimurium

A mutant of Salmonella typhimurium was found to be sensitive to killing by coliphage T7 because of an alteration in its surface properties. However, the infections were abortive and studies with (32)P-labeled T7 grown in Escherichia coli B (T7.B) indicated that the phage DNA was restricted by S. typhimurium. When a mutant T7 which survived the restriction and produced plaques on Salmonella (T7.S) was passed through one cycle of growth in E. coli B, its ability to grow in Salmonella was lost, indicating that host-controlled restriction and modification are operative in this system. Restrictionless S. typhimurium mutants were isolated that permit the growth of not only T7.S but also T7.B and coliphage T3. The physiology of T7 production in the restrictionless host is nearly identical to that in Escherichia coli.     

Growth of Salmonella typhimurium SL5319 and Escherichia coli F-18 in mouse cecal mucus: role of peptides and iron

Abstract Escherichia coli F-18, a normal human fecal isolate, and Salmonella typhimurium SL5319, an avirulent strain, are known to colonize the streptomycin-treated CD-1 mouse large intestine by utilizing nutrients present in intestinal mucus for growth. Moreover, previous experiments suggested the possibility that E. coli F-18 and S. typhimurium SL5319 utilized different mucus nutrients. Therefore, mouse cecal mucus was fractionated into high and low molecular weight components, and each fraction was inoculated either simultaneously or separately with E. coli F-18 and S. typhimurium SL5319. A 50 kd fraction was found in which the growth of S. typhimurium SL5319 suppressed growth of E. coli F-18. Evidence is presented that in this fraction S. typhimurium SL5319 utilizes peptides, presumably generated by mucus proteases, as a source of amino acids for growth. Furthermore, it is shown that S. typhimurium SL5319 grows in this 50 kd fraction with a generation time of 27 min in the presence of at most 7 μg of carbohydrate per ml and 2.2 μg of peptide per ml, and that S. typhimurium SL5319 suppresses E. coli F-18 growth in this fraction by sequestering iron. The data are discussed with respect to the role of peptide utilization and iron sequestration in the ability of S. typhimurium SL5319 to colonize the mouse large intestine.     

Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium.

We report the analysis of three open reading frames of Salmonella typhimurium LT2 which we identified as rfaF, the structural gene for ADP-heptose:LPS heptosyltransferase II; rfaD, the structural gene for ADP-L-glycero-D-manno-heptose-6-epimerase; and part of kbl, the structural gene for 2-amino-3-ketobutyrate CoA ligase. A plasmid carrying rfaF complements an rfaF mutant of S. typhimurium; rfaD and kbl are homologous to and in the same location as the equivalent genes in Escherichia coli K-12. The RfaF (heptosyl transferase II) protein shares regions of amino acid homology with RfaC (heptosyltransferase I), RfaQ (postulated to be heptosyltransferase III), and KdtA (ketodeoxyoctonate transferase), suggesting that these regions function in heptose binding. E. coli contains a block of DNA of about 1,200 bp between kbl and rfaD which is missing from S. typhimurium. This DNA includes yibB, which is an open reading frame of unknown function, and two promoters upstream of rfaD (P3, a heat-shock promoter, and P2). Both S. typhimurium and E. coli rfaD genes share a normal consensus promoter (P1). We postulate that the yibB segment is an insertion into the line leading to E. coli from the common ancestor of the two genera, though it could be a deletion from the line leading to S. typhimurium. The G+C content of the rfaLKZYJI genes of both S. typhimurium LT2 and E. coli K-12 is about 35%, much lower than the average of enteric bacteria; if this low G+C content is due to lateral transfer from a source of low G+C content, it must have occurred prior to evolutionary divergence of the two genera.     

Caenorhabditis elegans is a model host for Salmonella typhimurium

The idea of using simple, genetically tractable host organisms to study the virulence mechanisms of pathogens dates back at least to the work of Darmon and Depraitère [1]. They proposed using the predatory amoeba Dictyostelium discoideum as a model host, an approach that has proved to be valid in the case of the intracellular pathogen Legionella pneumophila [2]. Research from the Ausubel laboratory has clearly established the nematode Caenorhabditis elegans as an attractive model host for the study of Pseudomonas aeruginosa pathogenesis [3]. P. aeruginosa is a bacterium that is capable of infecting plants, insects and mammals. Other pathogens with a similarly broad host range have also been shown to infect C. elegans [3,4]. Nevertheless, the need to determine the universality of C. elegans as a model host, especially with regards pathogens that have a naturally restricted host specificity, has rightly been expressed [5]. We report here that the enterobacterium Salmonella typhimurium, generally considered to be a highly adapted pathogen with a narrow range of target hosts [6], is capable of infecting and killing C. elegans. Furthermore, mutant strains that exhibit a reduced virulence in mammals were also attenuated for their virulence in C. elegans, showing that the nematode may constitute a useful model system for the study of this important human pathogen.     

Nature of Lactose-fermenting Salmonella Strains Obtained from Clinical Sources

Six of seven lactose-fermenting (lac(+)) Salmonella strains obtained from clinical sources were found to be capable of transferring the lac(+) property by conjugation to Salmonella typhosa WR4204. All of the six S. typhosa strains which received the lac(+) property transferred it in turn to S. typhimurium WR5000 at the high frequencies typical of extrachromosomal F-merogenotes. These six lac elements were also transmissible from S. typhosa WR4204 to Proteus mirabilis and to some strains of Escherichia coli K-12; moreover, they were capable of promoting low frequency transfer of chromosomal genes from S. typhimurium WR5000 to S. typhosa WR4204. One of these lac elements was shown also to be capable of promoting low frequency chromosome transfer in E. coli K-12. E. coli K-12 strains harboring these lac elements exhibited sensitivity to the male specific phage R-17. Sensitivity to R-17 was not detected in Salmonella strains containing the elements. Examination of the lac elements in P. mirabilis by cesium chloride density gradient centrifugation showed that each element had a guanine plus cytosine content of 50%. The sizes of the elements varied from 0.8 to 3% of the total Proteus deoxyribonucleic acid. The amount of beta-galactosidase produced by induced and uninduced cultures of S. typhimurium WR5000 and S. typhosa WR4204 containing the lac elements was lower than that produced by these strains with the F-lac episome. The heat sensitivity of beta-galactosidase produced by the lac elements in their original Salmonella hosts indicated that the enzyme made by these strains differs from E. coli beta-galactosidase.     

Antimicrobial properties of the Escherichia coli R1 plasmid host killing peptide

The 52 amino acid host killing peptide (Hok) from the hok/sok post-segregational killer system of the Escherichia coli plasmid R1 was synthesized using Fmoc (9-fluorenylmethoxycarbonyl) chemistry, and its molecular weight was confirmed by mass spectroscopy. Hok kills cells by depolarizing the cytoplasmic membrane when it is made in the cytosol. Six microorganisms, E. coli, Bacillus subtilis, Pseudomonas aeruginosa, P. putida, Salmonella typhimurium, and Staphylococcus aureus were exposed to the purified peptide but showed no significant killing. However, electroporation of Hok (200 microgml(-1)) into E. coli cells showed a dramatic reduction (100000-fold) in the number of cells transformed with plasmid DNA which indicates that the synthetic Hok peptide killed cells. Electroporation of Hok into P. putida was also very effective with a 500-fold reduction in electrocompetent cells (100 microgml(-1)). Heat shock in the presence of Hok (380 microgml(-1)) resulted in a 5-fold reduction in E. coli cells but had no effect on B. subtilis. In addition, three Hok fragments (Hok(1-28), Hok(31-52) and Hok(16-52)) killed cells when electroporated into E. coli at 200 microgml(-1) (over 1000-fold killing for Hok(1-28), 50-fold killing for Hok(16-52) and over 1000-fold killing for Hok(31-52)). E. coli cells electroporated with Hok and visualized using transmission electron microscopy showed the same morphological changes as control cells to which Hok was induced using a plasmid inside the cell.