Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate ...in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection.
Carboxysomes are proteinaceous organelles that play essential roles in enhancing carbon fixation in cyanobacteria and some proteobacteria. These self-assembling organelles encapsulate Ribulose ...1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase using a protein shell structurally resembling an icosahedral viral capsid. The protein shell serves as a physical barrier to protect enzymes from the cytosol and a selectively permeable membrane to mediate transport of enzyme substrates and products. The structural and mechanical nature of native carboxysomes remain unclear. Here, we isolate functional β-carboxysomes from the cyanobacterium Synechococcus elongatus PCC7942 and perform the first characterization of the macromolecular architecture and inherent physical mechanics of single β-carboxysomes using electron microscopy, atomic force microscopy (AFM) and proteomics. Our results illustrate that the intact β-carboxysome comprises three structural domains, a single-layered icosahedral shell, an inner layer and paracrystalline arrays of interior Rubisco. We also observe the protein organization of the shell and partial β-carboxysomes that likely serve as the β-carboxysome assembly intermediates. Furthermore, the topography and intrinsic mechanics of functional β-carboxysomes are determined in native conditions using AFM and AFM-based nanoindentation, revealing the flexible organization and soft mechanical properties of β-carboxysomes compared to rigid viruses. Our study provides new insights into the natural characteristics of β-carboxysome organization and nanomechanics, which can be extended to diverse bacterial microcompartments and are important considerations for the design and engineering of functional carboxysomes in other organisms to supercharge photosynthesis. It offers an approach for inspecting the structural and mechanical features of synthetic metabolic organelles and protein scaffolds in bioengineering.
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.
The ST313 sequence type of Salmonella Typhimurium causes invasive non-typhoidal salmonellosis and was thought to be confined to sub-Saharan Africa. Two distinct phylogenetic lineages of African ST313 ...have been identified.
We analysed the whole genome sequences of S. Typhimurium isolates from UK patients that were generated following the introduction of routine whole-genome sequencing (WGS) of Salmonella enterica by Public Health England in 2014.
We found that 2.7% (84/3147) of S. Typhimurium from patients in England and Wales were ST313 and were associated with gastrointestinal infection. Phylogenetic analysis revealed novel diversity of ST313 that distinguished UK-linked gastrointestinal isolates from African-associated extra-intestinal isolates. The majority of genome degradation of African ST313 lineage 2 was conserved in the UK-ST313, but the African lineages carried a characteristic prophage and antibiotic resistance gene repertoire. These findings suggest that a strong selection pressure exists for certain horizontally acquired genetic elements in the African setting. One UK-isolated lineage 2 strain that probably originated in Kenya carried a chromosomally located bla
, demonstrating the continual evolution of this sequence type in Africa in response to widespread antibiotic usage.
The discovery of ST313 isolates responsible for gastroenteritis in the UK reveals new diversity in this important sequence type. This study highlights the power of routine WGS by public health agencies to make epidemiologically significant deductions that would be missed by conventional microbiological methods. We speculate that the niche specialisation of sub-Saharan African ST313 lineages is driven in part by the acquisition of accessory genome elements.
Over recent decades, Salmonella infection research has predominantly relied on murine infection models. However, in many cases the infection phenotypes of Salmonella pathovars in mice do not ...recapitulate human disease. For example, Salmonella Typhimurium ST313 is associated with enhanced invasive infection of immunocompromised people in Africa, but infection of mice and other animal models with ST313 have not consistently reproduced this invasive phenotype. The introduction of alternative infection models could help to improve the quality and reproducibility of pathogenesis research by facilitating larger-scale experiments. To investigate the virulence of S. Typhimurium ST313 in comparison with ST19, a combination of avian and insect disease models were used. We performed experimental infections in five lines of inbred and one line of outbred chickens, as well as in the alternative chick embryo and Galleria mellonella wax moth larvae models. This extensive set of experiments identified broadly similar patterns of disease caused by the African and global pathovariants of Salmonella Typhimurium in the chicken, the chicken embryo and insect models. A comprehensive analysis of all the chicken infection experiments revealed that the African ST313 isolate D23580 had a subtle phenotype of reduced levels of organ colonisation in inbred chickens, relative to ST19 strain 4/74. ST313 isolate D23580 also caused reduced mortality in chicken embryos and insect larvae, when compared with ST19 4/74. We conclude that these three infection models do not reproduce the characteristics of the systemic disease caused by S. Typhimurium ST313 in humans.
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.
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.
Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer
. However, numerous viruses and mobile ...genetic elements encode parts of the translational apparatus
, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature
. Our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.
Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally encoded cell surface ...structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 65 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the evolutionarily promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids.
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