Genetic variation in the MacAB-TolC efflux pump influences pathogenesis of invasive Salmonella isolates from Africa.

The various sub-species of Salmonella enterica cause a range of disease in human hosts. The human-adapted Salmonella enterica serovar Typhi enters the gastrointestinal tract and invades systemic sites to cause enteric (typhoid) fever. In contrast, most non-typhoidal serovars of Salmonella are primarily restricted to gut tissues. Across Africa, invasive non-typhoidal Salmonella (iNTS) have emerged with an ability to spread beyond the gastrointestinal tract and cause systemic bloodstream infections with increased morbidity and mortality. To investigate this evolution in pathogenesis, we compared the genomes of African iNTS isolates with other Salmonella enterica serovar Typhimurium and identified several macA and macB gene variants unique to African iNTS. MacAB forms a tripartite efflux pump with TolC and is implicated in Salmonella pathogenesis. We show that macAB transcription is upregulated during macrophage infection and after antimicrobial peptide exposure, with macAB transcription being supported by the PhoP/Q two-component system. Constitutive expression of macAB improves survival of Salmonella in the presence of the antimicrobial peptide C18G. Furthermore, these macAB variants affect replication in macrophages and influence fitness during colonization of the murine gastrointestinal tract. Importantly, the infection outcome resulting from these macAB variants depends upon both the Salmonella Typhimurium genetic background and the host gene Nramp1, an important determinant of innate resistance to intracellular bacterial infection. The variations we have identified in the MacAB-TolC efflux pump in African iNTS may reflect evolution within human host populations that are compromised in their ability to clear intracellular Salmonella infections.

The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid.

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These findings were consistent with the essentiality analyses of other S. Typhimurium ST19 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least one in vitro growth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our enzymological analysis revealed that the plasmid-encoded CysRSpBT1 had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of Gram-negative and Gram-positive bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated.

Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580.

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

Humanized nonobese diabetic-scid IL2rgammanull mice are susceptible to lethal Salmonella Typhi infection.

Salmonella enterica serovar Typhi, the cause of typhoid fever, is host-adapted to humans and unable to cause disease in mice. Here, we show that S. Typhi can replicate in vivo in nonobese diabetic (NOD)-scid IL2rgamma(null) mice engrafted with human hematopoietic stem cells (hu-SRC-SCID mice) to cause a lethal infection with pathological and inflammatory cytokine responses resembling human typhoid. In contrast, S. Typhi does not exhibit net replication or cause illness in nonengrafted or immunocompetent control animals. Screening of transposon pools in hu-SRC-SCID mice revealed both known and previously unknown Salmonella virulence determinants, including Salmonella Pathogenicity Islands 1, 2, 3, 4, and 6. Our observations indicate that the presence of human immune cells allows the in vivo replication of S. Typhi in mice. The hu-SRC-SCID mouse provides an unprecedented opportunity to gain insights into S. Typhi pathogenesis and devise strategies for the prevention of typhoid fever.

Mesophilic Aeromonas UDP-glucose pyrophosphorylase (GalU) mutants show two types of lipopolysaccharide structures and reduced virulence.

A mutation in galU that causes the lack of O34-antigen lipopolysaccharide (LPS) in Aeromonas hydrophila strain AH-3 was identified. It was proved that A. hydrophila GalU is a UDP-glucose pyrophosphorylase responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP. The galU mutant from this strain showed two types of LPS structures, represented by two bands on LPS gels. The first one (slow-migrating band in gels) corresponds to a rough strain having the complete core, with two significant differences: it lacks the terminal galactose residue from the LPS-core and 4-amino-4-deoxyarabinose residues from phosphate groups in lipid A. The second one (fast-migrating band in gels) corresponds to a deeply truncated structure with the LPS-core restricted to one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and three l-glycero-d-manno-heptose residues. galU mutants in several motile mesophilic Aeromonas strains from serotypes O1, O2, O11, O18, O21 and O44 were also devoid of the O-antigen LPS. The galU mutation reduced to less than 1 % the survival of these Aeromonas strains in serum, decreased the ability of these strains to adhere and reduced by 1.5 or 2 log units the virulence of Aeromonas serotype O34 strains in a septicaemia model in either fish or mice. All the changes observed in the galU mutants were rescued by the introduction of the corresponding single wild-type gene.

Analysis of the lateral flagellar gene system of Aeromonas hydrophila AH-3.

Mesophilic Aeromonas strains express a polar flagellum in all culture conditions, and certain strains produce lateral flagella on semisolid media or on surfaces. Although Aeromonas lateral flagella have been described as a colonization factor, little is known about their organization and expression. Here we characterized the complete lateral flagellar gene cluster of Aeromonas hydrophila AH-3 containing 38 genes, 9 of which (lafA-U) have been reported previously. Among the flgLL and lafA structural genes we found a modification accessory factor gene (maf-5) that is involved in formation of lateral flagella; this is the first time that such a gene has been described for lateral flagellar gene systems. All Aeromonas lateral flagellar genes were located in a unique chromosomal region, in contrast to Vibrio parahaemolyticus, in which the analogous genes are distributed in two different chromosomal regions. In A. hydrophila mutations in flhAL, lafK, fliJL, flgNL, flgEL, and maf-5 resulted in a loss of lateral flagella and reductions in adherence and biofilm formation, but they did not affect polar flagellum synthesis. Furthermore, we also cloned and sequenced the A. hydrophila AH-3 alternative sigma factor sigma54 (rpoN); mutation of this factor suggested that it is involved in expression of both types of flagella.

The UDP N-acetylgalactosamine 4-epimerase gene is essential for mesophilic Aeromonas hydrophila serotype O34 virulence.

Mesophilic Aeromonas hydrophila strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP N-acetylgalactosamine 4-epimerase (gne) confers the O(-) phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The gne gene is present in all the mesophilic Aeromonas strains tested. No changes were observed for the LPS core in a gne mutant from A. hydrophila strain AH-3 (serotype O34). O34 antigen LPS contains N-acetylgalactosamine, while no such sugar residue forms part of the LPS core from A. hydrophila AH-3. Some of the pathogenic features of A. hydrophila AH-3 gne mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the gne mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic Aeromonas strains, possess two kinds of flagella, and the absence of O34 antigen molecules by gne mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type gne gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).

Lateral flagella are required for increased cell adherence, invasion and biofilm formation by Aeromonas spp.

Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf- mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces.