Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation enhances its intracellular growth within macrophages

Modification of lipid A is essential for bacterial adaptation to its host. Salmonella Typhimurium LpxR potentially detoxifies lipid A by 3′-O-deacylation; however, the involvement of deacylation in its adaptation remains unclear. LpxR-dependent 3′-O-deacylation was observed in the stationary phase. When macrophages were infected with stationary phase bacteria, the intracellular growth of the lpxR-null strain was lower than that of the wild-type strain. Furthermore, the expression level of inducible nitric oxide synthase was higher in the cells infected with the lpxR-null strain than in the cells infected with the wild-type strain. These results indicate that lipid A 3′-O-deacylation is beneficial for intracellular growth.     

Structure of the O-polysaccharide of Salmonella enterica O41

An O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Salmonella enterica O41, and the following structure of the O-unit was determined by chemical analyses along with 1D and 2D ¹H and ¹³C NMR spectroscopy:→2)-β-d-Manp-(1→4)-α-d-Glcp-(1→3)-α-l-QuipNAc-(1→3)-α-d-GlcpNAc-(1→where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose. The structure established is in agreement with the O-antigen gene cluster of S. enterica O41 and tentative assignment of the gene functions reported earlier.     

Intracellular Demography and the Dynamics of Salmonella enterica Infections

An understanding of within-host dynamics of pathogen interactions with eukaryotic cells can shape the development of effective preventive measures and drug regimes. Such investigations have been hampered by the difficulty of identifying and observing directly, within live tissues, the multiple key variables that underlay infection processes. Fluorescence microscopy data on intracellular distributions of Salmonella enterica serovar Typhimurium (S. Typhimurium) show that, while the number of infected cells increases with time, the distribution of bacteria between cells is stationary (though highly skewed). Here, we report a simple model framework for the intensity of intracellular infection that links the quasi-stationary distribution of bacteria to bacterial and cellular demography. This enables us to reject the hypothesis that the skewed distribution is generated by intrinsic cellular heterogeneities, and to derive specific predictions on the within-cell dynamics of Salmonella division and host-cell lysis. For within-cell pathogens in general, we show that within-cell dynamics have implications across pathogen dynamics, evolution, and control, and we develop novel generic guidelines for the design of antibacterial combination therapies and the management of antibiotic resistance.     

Periplasmic disulfide isomerase DsbC is involved in the reduction of copper binding protein CueP from Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen with the ability to survive and replicate in macrophages. Periplasmic copper binding protein CueP is known to confer copper resistance to S. Typhimurium, and has been implicated in ROS scavenge activity by transferring the copper ion to a periplasmic superoxide dismutase or by directly reducing the copper ion. Structural and biochemical studies on CueP showed that its copper binding site is surrounded by conserved cysteine residues. Here, we present evidence that periplasmic disulfide isomerase DsbC plays a key role in maintaining CueP protein in the reduced state. We observed purified DsbC protein efficiently reduced the oxidized form of CueP, and that it acted on two (Cys104 and Cys172) of the three conserved cysteine residues. Furthermore, we found that a surface-exposed conserved phenylalanine residue in CueP was important for this process, which suggests that DsbC specifically recognizes the residue of CueP. An experiment using an Escherichia coli system confirmed the critical role played by DsbC in the ROS scavenge activity of CueP. Taken together, we propose a molecular insight into how CueP collaborates with the periplasmic disulfide reduction system in the pathogenesis of the bacteria.     

Mutations in the lipid A deacylase PagL which release the enzyme from its latency affect the ability of PagL to interact with lipopolysaccharide in Salmonella enterica serovar Typhimurium

PagL, a lipid A deacylase, is unique in that it is latent in the outer membrane of Salmonella enterica serovar Typhimurium. Several point mutations in the extracellular loops of PagL, which do not affect its enzymatic activity, release it from this latency. Precipitation analysis revealed that latent wild-type PagL associated with lipopolysaccharide, but non-latent PagL mutants did not. In contrast, non-latent PagL mutants preferentially associated with some membrane proteins. Precipitation analysis using inactive PagL mutants demonstrated that membrane lipid A deacylation did not affect association. These results indicate that mutations in the lipid A deacylase PagL which relieve the enzyme from its latency affect the ability of PagL to interact with lipopolysaccharide.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

The O-antigen of Salmonella enterica O13 and its relation to the O-antigen of Escherichia coli O127

The following structure of the O-polysaccharide (O-antigen) of Salmonella enterica O13 was established by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy:→2)-α-l-Fucp-(1→2)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen of S. enterica O13 was found to be closely related to that of Escherichia coli O127, which differs only in the presence of a GalNAc residue in place of the GlcNAc residue and O-acetylation. The location of the O-acetyl groups in the E. coli O127 polysaccharide was determined. The structures of the O-polysaccharides studied are in agreement with the DNA sequence of the O-antigen gene clusters of S. enterica O13 and E. coli O127 reported earlier.     

Development and validation of a resistance and virulence gene microarray targeting Escherichia coli and Salmonella enterica

A microarray was developed to simultaneously screen Escherichia coli and Salmonella enterica for multiple genetic traits. The final array included 203 60-mer oligonucleotide probes, including 117 for resistance genes, 16 for virulence genes, 25 for replicon markers, and 45 other markers. Validity of the array was tested by assessing inter-laboratory agreement among four collaborating groups using a blinded study design. Internal validation indicated that the assay was reliable (area under the receiver-operator characteristic curve = 0.97). Inter-laboratory agreement, however, was poor when estimated using the intraclass correlation coefficient, which ranged from 0.27 (95% confidence interval 0.24, 0.29) to 0.29 (0.23, 0.34). These findings suggest that extensive testing and procedure standardization will be needed before bacterial genotyping arrays can be readily shared between laboratories.     

Dissecting cobamide diversity through structural and functional analyses of the base-activating CobT enzyme of Salmonella enterica

Background

Cobamide diversity arises from the nature of the nucleotide base. Nicotinate mononucleotide (NaMN):base phosphoribosyltransferases (CobT) synthesize α-linked riboside monophosphates from diverse nucleotide base substrates (e.g., benzimidazoles, purines, phenolics) that are incorporated into cobamides.

Methods

Structural investigations of two members of the CobT family of enzymes in complex with various substrate bases as well as in vivo and vitro activity analyses of enzyme variants were performed to elucidate the roles of key amino acid residues important for substrate recognition.

Results

Results of in vitro and in vivo studies of active-site variants of the Salmonella enterica CobT (SeCobT) enzyme suggest that a catalytic base may not be required for catalysis. This idea is supported by the analyses of crystal structures that show that two glutamate residues function primarily to maintain an active conformation of the enzyme. In light of these findings, we propose that proper positioning of the substrates in the active site triggers the attack at the C1 ribose of NaMN.

Conclusion

Whether or not a catalytic base is needed for function is discussed within the framework of the in vitro analysis of the enzyme activity. Additionally, structure-guided site-directed mutagenesis of SeCobT broadened its substrate specificity to include phenolic bases, revealing likely evolutionary changes needed to increase cobamide diversity, and further supporting the proposed mechanism for the phosphoribosylation of phenolic substrates.

General Significance

Results of this study uncover key residues in the CobT enzyme that contribute to the diversity of cobamides in nature.     

Interaction of Candida albicans with an Intestinal Pathogen,Salmonella enterica Serovar Typhimurium

Candida albicans is an opportunistic human fungal pathogen that normally resides in the gastrointestinal tract and on the skin as a commensal but can cause life-threatening invasive disease. Salmonella enterica serovar Typhimurium is a gram-negative bacterial pathogen that causes a significant amount of gastrointestinal infection in humans. Both of these organisms are also pathogenic to the nematode Caenorhabditis elegans, causing a persistent gut infection leading to worm death. In the present study, we used a previously developed C. elegans polymicrobial infection model to assess the interactions between S. Typhimurium and C. albicans. We observed that when C. elegans is infected with C. albicans and serovar Typhimurium, C. albicans filamentation is inhibited. The inhibition of C. albicans filamentation by S. Typhimurium in C. elegans appeared to be mediated by a secretary molecule, since filter-sterilized bacterial supernatant was able to inhibit C. albicans filamentation. In vitro coculture assays under planktonic conditions showed that S. Typhimurium reduces the viability of C. albicans, with greater effects seen at 37°C than at 30°C. Interestingly, S. Typhimurium reduces the viability of both yeast and filamentous forms of C. albicans, but the killing appeared more rapid for the filamentous cells. The antagonistic interaction was also observed in a C. albicans biofilm environment. This study describes the interaction between two diverse human pathogens that reside within the gastrointestinal tract and shows that the prokaryote, S. Typhimurium, reduces the viability of the eukaryote, C. albicans. Identifying the molecular mechanisms of this interaction may provide important insights into microbial pathogenesis.Candida albicans, the most common human fungal pathogen, is a prototypical opportunistic organism that lives harmlessly in the human gastrointestinal tract but has the ability to cause life-threatening invasive disease. Bloodstream infection with C. albicans remains the most lethal form (10), with translocation of the gastrointestinal mucosa being an important pathogenic mechanism, especially in hemato-oncology patients and those who have undergone abdominal surgery. A key virulence determinant of C. albicans is its ability to transition from yeast to a filamentous form (16, 17, 19, 22). This morphogenesis appears important for tissue adherence and invasion (22). Furthermore, C. albicans has the ability to form complex biofilms on medical devices (13) and on human mucosal surfaces, such as the gastrointestinal and bronchial mucosa. C. albicans biofilm formation has immense clinical and economic consequences (13).Recently the interactions between this important fungal pathogen and bacteria were described (11, 12, 18). These studies focus on the interaction between C. albicans and nonfermenting, gram-negative bacteria, such as Pseudomonas aeruginosa and Acinetobacter baumannii, whose interactions are likely found in the clinical environment, especially in the respiratory tracts of critically ill patients and on wounds of patients with burn injuries (7, 20). Of interest, these bacteria show antagonistic properties toward C. albicans, with a predilection toward reducing the viability of C. albicans filaments. In order to study these prokaryote-eukaryote interactions, our laboratory developed a polymicrobial infection model system using Caenorhabditis elegans as a substitute host (18). Previously, we showed that C. albicans causes a persistent lethal infection of the C. elegans intestinal tract (6). This leads to overwhelming C. albicans intestinal proliferation with subsequent filamentation through the worm cuticle (6). Given these characteristics, we decided to use this model to study the interaction of C. albicans with another intestinal pathogen, Salmonella enterica serovar Typhimurium.S. Typhimurium is a gram-negative organism that belongs to the Enterobacteriaceae family. It is a gastrointestinal tract pathogen of humans, being responsible for approximately 2 million to 4 million cases of enterocolitis each year in the United States (4, 8, 21, 23). During infection, S. Typhimurium competes with normal intestinal flora (23). Its virulence pathways are well described, and it has been shown to cause a persistent and lethal gut infection of the nematode C. elegans, similar to infection seen with C. albicans (1, 14). Given this and the fact that C. albicans is a common inhabitant of the human gastrointestinal tract, we used the C. elegans polymicrobial infection model (18) to study the interactions between S. Typhimurium and C. albicans. Understanding the interactions between these diverse organisms within the complex milieu of an intestinal tract may provide important pathogenic and therapeutic insights.     

Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates

The accurate sub-typing of Salmonella enterica isolates is essential for epidemiological investigations and surveillance of Salmonella infections. Salmonella isolates are currently identified using the Kauffman-White serotyping scheme. Multilocus sequence typing (MLST) schemes have been developed for the major bacterial pathogens including Salmonella and have assisted in understanding the molecular epidemiology and population biology of these organisms. Recently, the DiversiLab rep-PCR system has been developed using micro-fluidic chips to provide standardized, semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 serovars, were analyzed using MLST and the DiversiLab rep-PCR system. MLST was able to identify 31 sequence types (STs), while the DiversiLab system revealed 38 DiversiLab types (DTs). The rep-PCR distinguished isolates of different serovars and showed greater discriminatory power (0.95) than MLST typing (0.89). Rep-PCR exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST typing with serotyping was 96%, representing a strong association. Comparison of rep-PCR profiles with those held in an online library database led to the accurate prediction of serovar in 63% of cases and resulted in inaccurate predictions for 10% of profiles. MLST and the rep-PCR system may provide useful additional informative techniques for the molecular identification of S. enterica. We conclude that the DiversiLab rep-PCR system may provide a rapid (less than 4 h) and standardized method for sub-typing isolates of S. enterica.     

The Salmonella enterica ZinT structure,zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc

Background

In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT–ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim.

Methods

ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy).

Results

The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a K_D value of 22 ± 2 nM for ZinT, while those with ZnuA point to one high affinity (K_D < 20 nM) and one low affinity Zn(II) binding site (K_D in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT–ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other.

Conclusions

ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA.

General significance

Knowledge of the ZinT–ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.     

Genetic Mechanisms Underlying the Pathogenicity of Cold-Stressed Salmonella enterica Serovar Typhimurium in Cultured Intestinal Epithelial Cells

Salmonella encounters various stresses in the environment and in the host during infection. The effects of cold (5°C, 48 h), peroxide (5 mM H₂O₂, 5 h) and acid stress (pH 4.0, 90 min) were tested on pathogenicity of Salmonella. Prior exposure of Salmonella to cold stress significantly (P < 0.05) increased adhesion and invasion of cultured intestinal epithelial (Caco-2) cells. This increased Salmonella-host cell association was also correlated with significant induction of several virulence-associated genes, implying an increased potential of cold-stressed Salmonella to cause an infection. In Caco-2 cells infected with cold-stressed Salmonella, genes involved in the electron transfer chain were significantly induced, but no simultaneous significant increase in expression of antioxidant genes that neutralize the effect of superoxide radicals or reactive oxygen species was observed. Increased production of caspase 9 and caspase 3/7 was confirmed during host cell infection with cold-stressed Salmonella. Further, a prophage gene, STM2699, induced in cold-stressed Salmonella and a spectrin gene, SPTAN1, induced in Salmonella-infected intestinal epithelial cells were found to have a significant contribution in increased adhesion and invasion of cold-stressed Salmonella in epithelial cells.     

DNA sequence of the araBAD-araC controlling region in Salmonella typhimurium LT2

The araB and araC genes of Salmonella typhimurium have been cloned onto the plasmid pBR322. Restriction analysis and subcloning of restriction fragments localized these genes to a 4.4 kb DNA fragment. Complementation analysis revealed that the cloned araB and araC genes from S. typhimurium complemented araB and araC mutant strains of Escherichia coli. Conversely, cloned araB and araC genes from E. coli complemented araB and araC mutant strains of S. typhimurium. The DNA sequences was determined for the S. typhimurium araB and araC controlling region and for the initially translated portions of these genes. The nucleotide sequence of the araB promoter was 87% homologous with the same region in E. coli and contained no deletions or insertions relative to the E. coli sequence. The presumed AUG codon corresponding to the amino terminus of the S. typhimurium araC protein was in the same location as in E. coli. There was, however, considerable divergence from the E. coli sequence preceding the translation start site. The nucleotide sequence of the initial 237 bp in the open reading frame of the S. typhimurium araC gene was 78% homologous with the same sequence in E. coli. By comparison, the amino acid sequence for this region was 91% conserved.     

Studies on the mechanism of Salmonella typhimurium enterotoxin-induced diarrhoea

The unidirectional fluxes of Na⁺ and Cl^? were studied in Salmonella typhimurium enterotoxin-treated rats. There was net secretion of Na⁺ and Cl^? in toxin-treated animals, while in control animals there was net absorption of these ions. In the presence of the Ca²⁺-ionophore, there was net secretion of Na²⁺ and Cl^? in the control group, while the ionophore enhanced the secretion of these ions in experimental anaimals. The calcium channel blocker, verapamil, decreased the secretion induced by salmonella toxin, but could not reverse the secretion of absorption. There was no difference in the net absorption of Ca²⁺ in both the control and experimental animals. There was a significant increase in the intracellular free calcium concentrations in enterocytes isolated from toxin-treated rat intestines as compared to that in enterocytes isolated from control animals. In the presence of PMA (phorobol-12-myristated-13-acetate) there was net secretion of Na⁺ and Cl^? in the control group, while in the experimental group there was no change in the fluxes of these ions. The selective, potent inhibitor of protein kinase C, H-7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine)_reversed the secretion of Na⁺ and Cl^? in the toxin-treated group to absorption. The addition of indomethacin also inhibited the secretion induced by salmonella toxin, but failed to reverse it to absorption. However, the addition both H-7 and indomethacin to the experimental group had a partial additive effect. These studies demonstrate that the Salmonella enterotoxin-mediated fluid secretion involved protein kinase C and the arachidonic acid metabolites and perhaps does not involve the extracellular calcium pools.