Injection of flagellin into the host cell cytosol by Salmonella enterica serotype Typhimurium

Bacterial flagellins are potent inducers of innate immunity. Three signaling pathways have been implicated in the sensing of flagellins; these involve toll-like receptor 5 (TLR5) and the cytosolic proteins Birc1e/Naip5 and Ipaf. Although the structural basis of TLR5-flagellin interaction is known, little is known about how flagellin enters the host cell cytosol to induce signaling via Birc1e/Naip5 and Ipaf. Here we demonstrate for the first time the translocation of bacterial flagellin into the cytosol of host macrophages by the vacuolar pathogen, Salmonella enterica serotype Typhimurium. Translocation of flagellin into the host cell cytosol was directly demonstrated using beta-lactamase reporter constructs. Flagellin translocation required the Salmonella Pathogenicity Island 1 Type III secretion system (SPI-1 T3SS) but not the flagellar T3SS.     

Inactivation of the flagellin gene of Salmonella enterica serotype enteritidis strongly reduces invasion into differentiated Caco-2 cells

A nonflagellated mutant of Salmonella enterica serotype Enteritidis was constructed by disrupting the flagellin gene (fliC). Northern blot analysis indicated that the mutation did not affect expression of the downstream fliU gene. Infection experiments with differentiated Caco-2 cells revealed that the mutant was about 50-fold less invasive than the wild-type strain, while bacterial adherence was unaffected. Complementation of the mutant with an intact fliC copy restored flagella formation and efficient bacterial invasion. Our data demonstrate that the fliC gene of S. enterica serotype Enteritidis is essential for the invasion of Caco-2 cells.     

Characterization of a T7-like lytic bacteriophage (phiSG-JL2) of Salmonella enterica serovar gallinarum biovar gallinarum

PhiSG-JL2 is a newly discovered lytic bacteriophage infecting Salmonella enterica serovar Gallinarum biovar Gallinarum but is nonlytic to a rough vaccine strain of serovar Gallinarum biovar Gallinarum (SG-9R), S. enterica serovar Enteritidis, S. enterica serovar Typhimurium, and S. enterica serovar Gallinarum biovar Pullorum. The phiSG-JL2 genome is 38,815 bp in length (GC content, 50.9%; 230-bp-long direct terminal repeats), and 55 putative genes may be transcribed from the same strand. Functions were assigned to 30 genes based on high amino acid similarity to known proteins. Most of the expected proteins except tail fiber (31.9%) and the overall organization of the genomes were similar to those of yersiniophage phiYeO3-12. phiSG-JL2 could be classified as a new T7-like virus and represents the first serovar Gallinarum biovar Gallinarum phage genome to be sequenced. On the basis of intraspecific ratios of nonsynonymous to synonymous nucleotide changes (Pi[a]/Pi[s]), gene 2 encoding the host RNA polymerase inhibitor displayed Darwinian positive selection. Pretreatment of chickens with phiSG-JL2 before intratracheal challenge with wild-type serovar Gallinarum biovar Gallinarum protected most birds from fowl typhoid. Therefore, phiSG-JL2 may be useful for the differentiation of serovar Gallinarum biovar Gallinarum from other Salmonella serotypes, prophylactic application in fowl typhoid control, and understanding of the vertical evolution of T7-like viruses.     

Sequence and structural analysis of the rfb (O antigen) gene cluster from a group C1 Salmonella enterica strain.

The rfb (O antigen) gene cluster of a group C1 Salmonella enterica strain was sequenced; it comprised seven open reading frames which precisely replaced the 16 open reading frames of a group B strain. Two genes of the mannose biosynthetic pathway were present: rfbK (phosphomannomutase) had a G+C content of 0.61 and had only 40% identity to rfbK of group B but was very similar to cpsG of the capsular polysaccharide pathway with 96% identity, whereas rfbM [guanosine diphosphomannose (GDP-Man) pyrophosphorylase] had a G+C content of 0.39. Other genes had G+C contents ranging from 0.24 to 0.28. rfbM(C1) and rfbM(B) had 60% identity, which is much less than expected within a species, but nonetheless indicates a much more recent common ancestor than for rfbK. The other genes showed much lower or no similarity to rfb genes of other S. enterica strains. It appears that the gene cluster evolved outside of Salmonella in a species with low G+C content: the rfbM gene presumably derives from that period whereas the rfbK gene appears to have arisen after transfer of the cluster to S. enterica by duplication of the S. enterica cpsG gene, presumably replacing an rfbK gene of low G+C content.     

Cloning and structure of group C1 O antigen (rfb gene cluster) from Salmonella enterica serovar montevideo.

The Salmonella enterica group C1 O antigen structure has a Man-Man-Man-Man-GlcNAc backbone with a glucose branch, which differs from the S. enterica group B O antigen structure which has a Man-Rha-Gal backbone with abequose as side-chain. We have cloned the group C1 rfb (O antigen) gene cluster from serovar montevideo strain M40, using a low-copy-number cosmid vector. The restriction map of the group C1 (M40) rfb gene cluster was compared with that of group B strain LT2 by Southern hybridization and restriction enzyme analysis. The results indicate that the flanking genes are very similar in the two strains, but there is no detectable similarity in the rfb regions. We localized the mannose pathway genes rfbM and rfbK and one of the genes, rfbK, shows considerably similarity to cpsG of strain LT2, suggesting that part of the mannose pathway in the group C1 rfb cluster is derived from a gene of the M antigen (cps) cluster. The M antigen, which forms a capsule, is comprised of four sugars, including fucose. The biosynthetic pathway of GDP-fucose has steps in common with the GDP-mannose pathway, and the cps cluster has isogenes of rfbK and rfbM, presumably as part of a fucose pathway. We discuss the structure and possible evolution of the group C1 rfb gene cluster.     

Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella typhimurium.

Previous studies of the phase 1 flagellar filament protein (flagellin) in strains of five serovars of Salmonella indicated that the central region of the fliC gene encoding the antigenic part of the protein is hypervariable both between and within serovars. To explore the possible use of this variation as a source of information on the phylogenetic relationships of closely related strains, we used the polymerase chain reaction technique to sequence part of the central region of the phase 1 flagellar genes of seven strains of Salmonella typhimurium that were known to differ in chromosomal genotype, as indexed by multilocus enzyme electrophoresis. We found that the nucleotide sequences of the central region were identical in all seven strains and determined that both the previously published sequence of the fliC gene in S. typhimurium LT2 and a report of a marked difference in the amino acid sequence of the phase 1 flagellins of two isolates of this serovar are erroneous. Our finding that the fliC gene is not evolving by sequence drift at an unusually rapid rate is compatible with a model that invokes lateral transfer and recombination of the flagellin genes as a major evolutionary process generating new serovars (antigen combinations) of salmonellae.     

Genotypes of multidrug-resistant Salmonella enterica serotype typhimurium from two regions of Kenya

A combination of phage typing and pulsed-field gel electrophoresis of Xbal-digested chromosomal DNA has been used to study the epidemiological relationships of multidrug-resistant Salmonella enterica serotype typhimurium from Nairobi (64 isolates) and Kilifi (40 isolates) collected over the period 1994-1997. Isolates from Nairobi belonged to 11 definitive phage types (DTs) encompassing eight different PFGE patterns. In contrast, isolates from Kilifi were mainly DT 56 (60%) and all fell into a single PFGE pattern. The remaining isolates did not conform to a recognisable phage type. We conclude that multidrug-resistant S. typhimurium infections from Nairobi were caused by multiple strains while those from Kilifi were likely to be from a microepidemic caused by a single clone.     

Microarray-based analysis of IncA/C plasmid-associated genes from multidrug-resistant Salmonella enterica

In the family Enterobacteriaceae, plasmids have been classified according to 27 incompatibility (Inc) or replicon types that are based on the inability of different plasmids with the same replication mechanism to coexist in the same cell. Certain replicon types such as IncA/C are associated with multidrug resistance (MDR). We developed a microarray that contains 286 unique 70-mer oligonucleotide probes based on sequences from five IncA/C plasmids: pYR1 (Yersinia ruckeri), pPIP1202 (Yersinia pestis), pP99-018 (Photobacterium damselae), pSN254 (Salmonella enterica serovar Newport), and pP91278 (Photobacterium damselae). DNA from 59 Salmonella enterica isolates was hybridized to the microarray and analyzed for the presence or absence of genes. These isolates represented 17 serovars from 14 different animal hosts and from different geographical regions in the United States. Qualitative cluster analysis was performed using CLUSTER 3.0 to group microarray hybridization results. We found that IncA/C plasmids occurred in two lineages distinguished by a major insertion-deletion (indel) region that contains genes encoding mostly hypothetical proteins. The most variable genes were represented by transposon-associated genes as well as four antimicrobial resistance genes (aphA, merP, merA, and aadA). Sixteen mercury resistance genes were identified and highly conserved, suggesting that mercury ion-related exposure is a stronger pressure than anticipated. We used these data to construct a core IncA/C genome and an accessory genome. The results of our studies suggest that the transfer of antimicrobial resistance determinants by transfer of IncA/C plasmids is somewhat less common than exchange within the plasmids orchestrated by transposable elements, such as transposons, integrating and conjugative elements (ICEs), and insertion sequence common regions (ISCRs), and thus pose less opportunity for exchange of antimicrobial resistance.     

Recombination of Salmonella phase 1 flagellin genes generates new serovars.

To determine the evolutionary mechanisms generating serotypic diversity in Salmonella strains, we sequenced the central, antigen-determining part of the phase 1 flagellin gene (fliC) in strains of several serovars for which estimates of chromosomal genomic relatedness had been obtained by multilocus enzyme electrophoresis. The nucleotide sequence of this region was identical in several chromosomally divergent strains of Salmonella heidelberg (phase 1 antigen r) but differed by 19% from the corresponding and similarly invariant sequence in strains of the closely related serovar Salmonella typhimurium (phase 1 antigen i). Mutational drift of the sequence present in the common ancestor is unlikely to have generated the difference between the phase 1 flagellins of these two serovars, which we attribute instead to a recombination event. This interpretation is supported by evidence that Salmonella strains of very diverse chromosomal backgrounds but similar phase 1 antigens may have closely similar nucleotide sequences for this highly polymorphic region. We suggest that lateral transfer and recombination of phase 1 flagellin genes is a major evolutionary mechanism generating new Salmonella serovars.     

Comparison of Salmonella enterica serotype Infantis isolates from a veterinary teaching hospital

AIMS: To compare Salmonella enterica serotype Infantis isolates obtained from patients or the environment of a veterinary teaching hospital over a period of 9 years following a nosocomial outbreak to determine whether isolates were epidemiologically related or represented unrelated introductions into the hospital environment. METHODS AND RESULTS: Fifty-six S. Infantis isolates were compared based on their phenotypic (antimicrobial drug [AMD] susceptibility pattern) and genotypic (pulsed-field gel electrophoresis [PFGE] pattern and presence of integrons) characteristics. Epidemiologically unrelated S. Infantis isolates clustered separately from all but two of the hospital isolates, and several isolates from different years and various sources were indistinguishable from each other in cluster analysis of two-enzyme PFGE results. A high percentage of isolates (80.3%) were resistant to at least one AMD, with 67.8% showing resistance to >5 AMD. The majority (74.1%) of isolates tested contained type 1 integrons. CONCLUSION: Results strongly suggest that there was nosocomial transmission of S. Infantis during the initial outbreak, and that contamination arising from this outbreak persisted across years despite rigorous hygiene and biosecurity precautions and may have led to subsequent nosocomial infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence of persistence and transmission of Salmonella clones across years, even in the face of rigorous preventive measures, has important implications for other facilities that have experienced outbreaks of Salmonella infections.     

Translation inhibition of the Salmonella fliC gene by the fliC 5' untranslated region, fliC coding sequences, and FlgM

The 5'-untranslated region (5'UTR) of the fliC flagellin gene of Salmonella contains sequences critical for efficient fliC mRNA translation coupled to assembly. In a previous study we used targeted mutagenesis of the 5' end of the fliC gene to isolate single base changes defective in fliC gene translation. This identified a predicted stem-loop structure, SL2, as an effector of normal fliC mRNA translation. A single base change (-38C:U) in the fliC 5'UTR resulted in a mutant that is defective in fliC mRNA translation and was chosen for this study. Motile (Mot+) revertants of the -38C:T mutant were isolated and characterized, yielding several unexpected results. Second-site suppressors that restored fliC translation and motility included mutations that disrupt a RNA duplex stem formed between RNA sequences in the fliC 5'UTR SL2 region (including a precise deletion of SL2) and bases early within the fliC-coding region. A stop codon mutation at position 80 of flgM also suppressed the -38C:T motility defect, while flgM mutants defective in anti-sigma28 activity had no effect on fliC translation. One remarkable mutation in the fliC 5'UTR (-15G:A) results in a translation defect by itself but, in combination with the -38C:U mutation, restores normal translation. These results suggests signals intrinsic to the fliC mRNA that have both positive and negative effects on fliC translation involving both RNA structure and interacting proteins.     

Sequence analysis of operator mutants of the phase-1 flagellin-encoding gene, fliC, in Salmonella typhimurium

In phase-2 cells of diphasic Salmonella strains, expression of the phase-1 flagellin-encoding gene, fliC, is repressed by the repressor encoded by the fljA gene. Nine operator-constitutive (Oc) mutants of fliC were isolated from S. typhimurium by selecting those which could express fliC in the presence of the repressor. Among them, eight mutants could express fliC both in the presence and the absence of the repressor, whereas the ninth one could express only in the presence of the repressor. Nucleotide sequence analysis revealed that the Oc mutations of the former type were all located between bp 7 and 20 upstream from the coding region of fliC, which suggests that this region may correspond to the operator for fliC. The latter mutant was found to have a tandem duplication of 28 bp which contains a part of the operator sequence, and seems to require the repressor to activate fliC expression.     

Ferrioxamine-mediated Iron(III) utilization by Salmonella enterica

Utilization of ferrioxamines as sole sources of iron distinguishes Salmonella enterica serotypes Typhimurium and Enteritidis from a number of related species, including Escherichia coli. Ferrioxamine supplements have therefore been used in preenrichment and selection media to increase the bacterial growth rate while selectivity is maintained. We characterized the determinants involved in utilization of ferrioxamines B, E, and G by S. enterica serotype Typhimurium by performing siderophore cross-feeding bioassays. Transport of all three ferric siderophores across the outer membrane was dependent on the FoxA receptor encoded by the Fur-repressible foxA gene. However, only the transport of ferrioxamine G was dependent on the energy-transducing protein TonB, since growth stimulation of a tonB strain by ferrioxamines B and E was observed, albeit at lower efficiencies than in the parental strain. Transport across the inner membrane was dependent on the periplasmic binding protein-dependent ABC transporter complex comprising FhuBCD, as has been reported for other hydroxamate siderophores of enteric bacteria. The distribution of the foxA gene in the genus Salmonella, as indicated by DNA hybridization studies and correlated with the ability to utilize ferrioxamine E, was restricted to subspecies I, II, and IIIb, and this gene was absent from subspecies IIIa, IV, VI, and VII (formerly subspecies IV) and Salmonella bongori (formerly subspecies V). S. enterica serotype Typhimurium mutants with either a transposon insertion or a defined nonpolar frameshift (+2) mutation in the foxA gene were not able to utilize any of the three ferrioxamines tested. A strain carrying the nonpolar foxA mutation exhibited a significantly reduced ability to colonize rabbit ileal loops compared to the foxA+ parent. In addition, a foxA mutant was markedly attenuated in mice inoculated by either the intragastric or intravenous route. Mice inoculated with the foxA mutant were protected against subsequent challenge by the foxA+ parent strain.     

Genetic diversity of multi-resistant Salmonella enterica serotype Typhimurium isolates from animals and humans

In this study, the genetic diversities of multi-resistant Salmonella typhimurium (ST) isolates were analyzed via the application of both pulsed field gel electrophoresis (PFGE) and Polymerase chain reaction (PCR) analysis methods, using 6 kinds of primers (REP, ERIC, SERE, BOX, P-1254 and OPB-17). And their discriminative abilities (DA) were also compared in order to determine the most effective and reliable analysis method. 118 S. typhimurium isolates, cultured from diverse animals and human patients in Korea beginning in 1993, were analyzed and subjected to a comparison of Simpson's index of diversity (SID), using both PFGE and PCR methods. PFGE by XbaI enzyme digestion allowed for discrimination into 9 pulsotypes, with high SID values (0.991) on the genomic DNA level. This shows that PFGE is a very discriminative genotypic tool, and also that multiple clones of S. typhimurium isolates had existed in domestic animals and humans in Korea since 1993. However, we could ultimately not to trace the definitive sources or animal reservoirs of specific S. typhimurium isolates examined in this study. Depending on the SID values, the combined method (7 kinds of method) was found to be the most discriminative method, followed by (in order) SERE-PCR, REP-PCR, ERIC-PCR, PFGE & OPB-17 (RAPD), P-1254 (RAPD), and BOX-PCR at the 80% clone cut-off value. This finding suggests that the REP-PCR method (which utilizes 4 primer types) may be an alternative tool to PFGE for the genotyping of S. typhimurium isolates, with comparable cost, time, and labor requirement. The establishment of a highly reliable and discriminatory method for epidemiologic analysis is considered necessary in order for researchers to trace the sources of specific pathogens and, consequently, to control and prevent the spread of epidemic S. typhimurium isolates to humans.     

Population heterogeneity of Salmonella enterica serotype Typhimurium resulting from phase variation of the lpf operon in vitro and in vivo

The lpf fimbrial operon oscillates between phase ON and phase OFF expression states, thereby generating heterogeneity within S. enterica serotype Typhimurium populations with regard to expression of long polar fimbrial antigens. To determine whether the proportion of lpf phase variants changes with growth conditions, the lpf phase ON content of cultures was determined after in vitro and in vivo passage. After passage in Luria-Bertani (LB) broth for 120 generations, 96% of cells in a serotype Typhimurium culture carried the lpf operon in the phase ON expression state, regardless of the phase ON/OFF ratio in the inoculum. In contrast, a culture passaged on LB agar plates for 500 generations contained approximately 2% lpf phase ON cells. Differences in the lpf phase ON content of cultures passaged in broth and on plates were not caused by an outgrowth of lpf phase ON or lpf phase OFF cells, since deletion of lpf biosynthesis genes did not alter the phase ON/OFF ratio attained after passage. Instead, growth in LB broth resulted in a eightfold increase in the phase OFF-to-ON transition frequency and a decrease of the lpf phase ON-to-OFF transition frequency by a factor of 150 compared to growth on LB agar plates. After infection of na?ve CBA/J mice with an lpf phase ON culture of serotype Typhimurium, the proportion of lpf phase ON cells continuously decreased over time, regardless of whether the strain carried intact fimbrial biosynthesis genes. These data suggest that elaboration of fimbriae does not have a major influence on the population heterogeneity produced by phase variation of the lpf operon in na?ve mice.     

Survival kinetics of Salmonella enterica serotype senftenberg (S. senftenberg) after heat and acid stress

The survival kinetics of two clinical isolates of Salmonella senftenberg were studied after heat and acid stress. The strains survived better at 53 and 55 °C after heat shock of 30 min at 50 °C or overnight heat adaptation at 45 °C. An increase in the decimal reduction time, D, of heat-shocked [10.2 min (53 °C) and 9.37 min (55 °C)] and heat-adapted [8.12 min (53 °C) and 7.8 min (55 °C)] cells was observed compared with the non-stressed cells [6.87 min (53 °C), 6.56 min 55 °C)]. A significant difference was also observed in the survival of acid-adapted to acid non-adapted S. senftenberg bacteria.