The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP(CT)) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP(CT) allowing us to obtain highly purified protein. We demonstrate that WbaP(CT) is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP(CT) domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.

Type IVB pili are required for invasion but not for adhesion of Salmonella enterica serovar Typhi into BHK epithelial cells in a cystic fibrosis transmembrane conductance regulator-independent manner

The cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed as an epithelial cell receptor for the entry of Salmonella Typhi but not Salmonella Typhimurium. The bacterial ligand recognized by CM is thought to reside either in the S. Typhi lipopolysaccharide core region or in the type IV pili. Here, we assessed the ability of virulent strains of S. Typhi and S. Typhimurium to adhere to and invade BHK epithelial cells expressing either the wild-type CFTR protein or the Delta F508 CFTR mutant. Both S. Typhi and S. Typhimurium invaded the epithelial cells in a CFTR-independent fashion. Furthermore and also in a CFTR-independent manner, a S. Typhi pilS mutant adhered normally to BHK cells but displayed a 50% reduction in invasion as compared to wild-type bacteria. Immunofluorescence microscopy revealed that bacteria and CFTR do not colocalize at the epithelial cell surface. Together, our results strongly argue against the established dogma that CFTR is a receptor for entry of Salmonella to epithelial cells. (C) 2011 Elsevier Ltd. All rights reserved.

Functional analysis of the C-terminal domain of the WbaP protein that mediates initiation of O antigen synthesis in Salmonella enterica

WbaP catalyzes the transfer of galactose-1-phosphate onto undecaprenyl phosphate (Und-P). The enzyme belongs to a large family of bacterial membrane proteins required for initiation of the synthesis of O antigen lipopolysaccharide and polysaccharide capsules. Previous work in our laboratory demonstrated that the last transmembrane helix and C-terminal tail region of WbaP (WbaP(CT)) are sufficient for enzymatic activity. Here, we demonstrate the cytoplasmic location of the WbaP C-terminal tail and show that WbaPCT domain N-terminally fused to thioredoxin (TrxA-WbaP(CT)) exhibits improved protein folding and enhanced transferase activity. Alanine replacement of highly conserved charged or polar amino acids identified seven critical residues for enzyme activity in vivo and in vitro. Four of these residues are located in regions predicted to be a-helical. These regions and their secondary structure predictions are conserved in distinct WbaP family members, suggesting they may contribute to form a conserved catalytic center.

Dam methylation controls O-antigen chain length in Salmonella enterica serovar enteritidis by regulating the expression of Wzz protein

We reported previously that a Salmonella enterica serovar Enteritidis dam mutant expressing a truncated Dam protein does not agglutinate in the presence of specific antibodies against O9 polysaccharide. Here we investigate the participation of Dam in lipopolysaccharide (LPS) synthesis in Salmonella. The LPS O-antigen profiles of a dam null mutant (SEDeltadam) and the Salmonella serovar Enteritidis parental strain were examined by using electrophoresis and silver staining. Compared to the parental strain, SEDeltadam produced LPS with shorter O-antigen polysaccharide chains. Since Wzz is responsible for the chain length distribution of the O antigen, we investigated whether Dam methylation is involved in regulating wzz expression. Densitometry analysis showed that the amount of Wzz produced by SEDeltadam is threefold lower than the amount of Wzz produced by the parental strain. Concomitantly, the activity of the wzz promoter in SEDeltadam was reduced nearly 50% in logarithmic phase and 25% in stationary phase. These results were further confirmed by reverse transcription-PCR showing that wzz gene expression was threefold lower in the dam mutant than in the parental strain. Our results demonstrate that wzz gene expression is downregulated in a dam mutant, indicating that Dam methylation activates expression of this gene. This work indicates that wzz is a new target regulated by Dam methylation and demonstrates that DNA methylation not only affects the production of bacterial surface proteins but also the production of surface polysaccharides.

The outer core lipopolysaccharide of Salmonella enterica serovar Typhi is required for bacterial entry into epithelial cells

Salmonella enterica serovar Typhi causes typhoid fever in humans. Central to the pathogenicity of serovar Typhi is its capacity to invade intestinal epithelial cells. The role of lipopolysaccharide (LPS) in the invasion process of serovar Typhi is unclear. In this work, we constructed a series of mutants with defined deletions in genes for the synthesis and polymerization of the O antigen (wbaP, wzy, and wzz) and the assembly of the outer core (waaK, waaJ, waaI, waaB, and waaG). The abilities of each mutant to associate with and enter HEp-2 cells and the importance of the O antigen in serum resistance of serovar Typhi were investigated. We demonstrate here that the presence and proper chain length distribution of the O-antigen polysaccharide are essential for serum resistance but not for invasion of epithelial cells. In contrast, the outer core oligosaccharide structure is required for serovar Typhi internalization in HEp-2 cells. We also show that the outer core terminal glucose residue (Glc II) is necessary for efficient entry of serovar Typhi into epithelial cells. The Glc I residue, when it becomes terminal due to a polar insertion in the waaB gene affecting the assembly of the remaining outer core residues, can partially substitute for Glc II to mediate bacterial entry into epithelial cells. Therefore, we conclude that a terminal glucose in the LPS core is a critical residue for bacterial recognition and internalization by epithelial cells.

RpoS and RpoN are involved in the growth-dependent regulation of rfaH transcription and O antigen expression in Salmonella enterica serovar Typhi

We reported earlier that the production of O antigen lipopolysaccharide (LPS) by Salmonella enterica serovar Typhi (Salmonella typhi) increases at the onset of stationary phase and correlates with a growth-regulated expression of the rfaH gene under the control of the alternative sigma factor RpoN (Microbiology 148 (2002) 3789). In this study, we demonstrate that RpoS also modulates rfaH promoter activity as revealed by the absence of growth-dependent regulation of an rfaH-lacZ transcriptional fusion and O antigen production in a S. typhi rpoS mutant. Introduction of a constitutively expressed rpoN gene into the rpoS mutant restored increased production of O antigen during stationary phase, suggesting that constitutive production of RpoN could overcome the RpoS defect. Similar results were observed when an rpoS rpoN double mutant was transformed with the intact rpoN gene. Thus, we conclude that both RpoS and RpoN control the rfaH promoter activity and concomitantly, the production of O-specific LPS in S. typhi.

Tigecycline challenge triggers sRNA production in Salmonella enterica serovar Typhimurium

Background: Bacteria employ complex transcriptional networks involving multiple genes in response to stress, which is not limited to gene and protein networks but now includes small RNAs (sRNAs). These regulatory RNA molecules are increasingly shown to be able to initiate regulatory cascades and modulate the expression of multiple genes that are involved in or required for survival under environmental challenge. Despite mounting evidence for the importance of sRNAs in stress response, their role upon antibiotic exposure remains unknown. In this study, we sought to determine firstly, whether differential expression of sRNAs occurs upon antibiotic exposure and secondly, whether these sRNAs could be attributed to microbial tolerance to antibiotics.

Results: A small scale sRNA cloning strategy of Salmonella enterica serovar Typhimurium SL1344 challenged with half the minimal inhibitory concentration of tigecycline identified four sRNAs (sYJ5, sYJ20, sYJ75 and sYJ118) which were reproducibly upregulated in the presence of either tigecycline or tetracycline. The coding sequences of the four sRNAs were found to be conserved across a number of species. Genome analysis found that sYJ5 and sYJ118 mapped between the 16S and 23S rRNA encoding genes. sYJ20 (also known as SroA) is encoded upstream of the tbpAyabKyabJ operon and is classed as a riboswitch, whilst its role in antibiotic stress-response appears independent of its riboswitch function. sYJ75 is encoded between genes that are involved in enterobactin transport and metabolism. Additionally we find that the genetic deletion of sYJ20 rendered a reduced viability phenotype in the presence of tigecycline, which was recovered when complemented. The upregulation of some of these sRNAs were also observed when S. Typhimurium was challenged by ampicillin (sYJ5, 75 and 118); or when Klebsiella pneumoniae was challenged by tigecycline (sYJ20 and 118).

Conclusions: Small RNAs are overexpressed as a result of antibiotic exposure in S. Typhimurium where the same molecules are upregulated in a related species or after exposure to different antibiotics. sYJ20, a riboswitch, appears to possess a trans-regulatory sRNA role in antibiotic tolerance. These findings imply that the sRNA mediated response is a component of the bacterial response to antibiotic challenge.

O-antigen expression in Salmonella enterica serovar Typhi is regulated by nitrogen availability through RpoN-mediated transcriptional control of the rfaH gene

The authors previously reported increased expression of the Salmonella enterica serovar Typhi (S. typhi) rfaH gene when the bacterial cells reach stationary phase. In this study, using a lacZ fusion to the rfaH promoter region, they demonstrate that growth-dependent regulation of rfaH expression occurs at the level of transcription initiation. It was also observed that production of the lipopolysaccharide (LPS) O-antigen by S. typhi Ty2 correlated with the differential expression of rfaH during bacterial growth. This was probably due to the increased cellular levels of RfaH, since expression of the distal gene in the O-antigen gene cluster of S. typhi Ty2, wbaP, was also increased during stationary growth, as demonstrated by RT-PCR analysis. Examination of the sequences upstream of the rfaH coding region revealed homologies to potential binding sites for the RcsB/RcsA dimer of the RcsC/YopJ/RcsB phosphorelay regulatory system and for the RpoN alternative sigma factor. The expression of the rfaH gene in rpoN and rcsB mutants of S. typhi Ty2 was measured. The results indicate that inactivation of rpoN, but not of rcsB, suppresses the growth-phase-dependent induction of rfaH expression. Furthermore, production of beta-galactosidase mediated by the rfaH-lacZ fusion increased approximately fourfold when bacteria were grown in a nitrogen-limited medium. Nitrogen limitation was also shown to increase the expression of the O-antigen by the wild-type S. typhi Ty2, as demonstrated by a similar electrophoretic profile to that observed during the stationary phase of growth in rich media. It is therefore concluded that the relationship between LPS production and nitrogen limitation parallels the pattern of rfaH regulation under the control of RpoN and is consistent with the idea that RpoN modulates LPS formation via its effect on rfaH gene expression during bacterial growth.

Production and evaluation of the utility of novel phage display-derived peptide ligands to Salmonella spp. for magnetic separation

Aims: The objectives of this study were to produce Salmonella-specific peptide ligands by phage display biopanning and evaluate their use for magnetic separation (MS).
Methods and Results: Four phage display biopanning rounds were performed and the peptides expressed by the two most Salmonella-specific (on the basis of phage binding ELISA results) phage clones, MSal020401 and MSal020417, were chemically synthesized and coupled to MyOne™ tosylactivated Dynabeads®. Peptide capture capability for whole Salmonella cells from non-enriched broth cultures was quantified by MS + plate counts and MS + Greenlight™ detection, and compared to capture capability of anti-Salmonella (antibody-coated) Dynabeads®. MS + Greenlight™ gave a more comprehensive picture of capture capability than MS + plate counts and showed that Peptide MSal020417-coated beads exhibited at least similar, if not better, capture capability to anti-Salmonella Dynabeads® (mean capture values of 36.0 ± 18.2 % and 31.2 ± 20.1 %, respectively, over Salmonella spp. concentration range 3 x 101 - 3 x 106 cfu ml-1) with minimal cross-reactivity (= 1.9 %) to three other foodborne bacteria.
Conclusions: One of the phage display-derived peptide ligands was demonstrated by MS + Greenlight™ to be a viable antibody-alternative for MS of Salmonella spp.
Significance and Impact of Study: This study demonstrates an antibody-free approach to Salmonella detection and opens substantial possibilities for more rapid tests for this bacterium.

Multiplex Solid-Phase PCR for Rapid Detection and Identification of Salmonella spp. at Sub-species

This study presents a solid-phase PCR (SP-PCR) for rapid detection, identification, and sub-typing of various Salmonella species, the major food-borne cause of salmonellosis. The target DNA is firstly amplified with PCR primers (one primer is labeled with fluorophores) in the liquid phase. Simultaneously on the solid phase, the amplified PCR amplicons interact with the nested DNA probes immobilized on the solid substrate as an array. If the immobilized probes match the sequence of the DNA templates they are extended by the polymerase and serve as template for the second strand elongation primed by the liquid phase primer thus generating new templates for the SP-PCR. After the reaction, PCR products labeled with fluorophores remain attached to the substrate and can be visualized directly by fluorescence readout devices. Using this method, S. enteritidis, S. typhimurium and S. dublin can be detected at the same time. The method offers several advantages over conventional multiplex PCR: less competition between different primer pairs thus increasing multiplexing capability, only single wavelength optical readout needed for the multiplexing detection, and less time-consuming owing to reduction of the post-PCR gel electrophoresis. The method will be useful for development of point-of-care devices for rapid detection and identification of Salmonella spp. A solid-phase PCR for rapid detection and identification of S. enteritidis, S. typhimurium and S. dublin is developed. The method offers advantages such as better multiplexing capability, only single wavelength optical readout needed, and less time-consuming.

Efficacy of Biocides Used in the Modern Food Industry To Control Salmonella enterica, and Links between Biocide Tolerance and Resistance to Clinically Relevant Antimicrobial Compounds

Biocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR) Salmonella enterica strains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds of in vitro selection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure of Salmonella strains to an active biocidal compound, a high-level of tolerance was selected for a number of Salmonella serotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonic Salmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.

ProP Is Required for the Survival of Desiccated Salmonella enterica Serovar Typimurium Cells on a Stainless Steel Surface

Consumers trust commercial food production to be safe, and it is important to strive to improve food safety at every level. Several outbreaks of food-borne disease have been caused by Salmonella strains associated with dried food. Currently we do not know the mechanisms used by Salmonella enterica serovar Typhimurium to survive in desiccated environments. The aim of this study was to discover the responses of S. Typhimurium ST4/74 at the transcriptional level to desiccation on a stainless steel surface and to subsequent rehydration. Bacterial cells were dried onto the same steel surfaces used during the production of dry foods, and RNA was recovered for transcriptomic analysis. Subsequently, dried cells were rehydrated and were again used for transcriptomic analysis. A total of 266 genes were differentially expressed under desiccation stress compared with a static broth culture. The osmoprotectant transporters proP, proU, and osmU (STM1491 to STM1494) were highly upregulated by drying. Deletion of any one of these transport systems resulted in a reduction in the long-term viability of S. Typhimurium on a stainless steel food contact surface. The proP gene was critical for survival; proP deletion mutants could not survive desiccation for long periods and were undetectable after 4 weeks. Following rehydration, 138 genes were differentially expressed, with upregulation observed for genes such as proP, proU, and the phosphate transport genes (pstACS). In time, this knowledge should prove valuable for understanding the underlying mechanisms involved in pathogen survival and should lead to improved methods for control to ensure the safety of intermediate-and low-moisture foods. © 2013, American Society for Microbiology.

Development of M13 bacteriophage-based SPR detection using Salmonella detection as a case study

Surface plasmon resonance (SPR)-based biosensor is a popular platform for real-time monitoring and sensitive detection for a myriad of targets. However, only a few studies have reported the use of bacteriophages as specific binders for SPR-based detection. This study aimed to demonstrate how filamentous M13 bacteriophages expressing 12-mer peptides can be employed in an SPR-based assay, using a Salmonella-specific bacteriophage as a model binder to detect the foodborne bacterium Salmonella. Several important factors (immobilization buffers and methods, and interaction buffers) for a successful bacteriophage-based SPR assay were optimized. As a result, a Salmonella-specific bacteriophage-based SPR assay was achieved, with very low cross reactivity with other non-target foodborne pathogens and detection limits of 8.0 × 10⁷ and 1.3 × 10⁷ CFU/mL for one-time and five-time immobilized sensors, respectively. This proof-of-concept study demonstrates the feasibility of using M13 bacteriophages expressing target-specific peptides as a binder in a rapid and label-free SPR assay for pathogen detection.