Genes Required for the Fitness of Salmonella enterica Serovar Typhimurium during Infection of Immunodeficient gp91 (cid:2) / (cid:2) phox Mice

Salmonella enterica causes systemic diseases (typhoid and paratyphoid fever), nontyphoidal septicemia (NTS), and gastroenteri-tis in humans and other animals worldwide. An important but underrecognized emerging infectious disease problem in sub-Saharan Africa is NTS in children and immunocompromised adults. A current goal is to identify Salmonella mutants that are not pathogenic in the absence of key components of the immune system such as might be found in immunocompromised hosts. Such attenuated strains have the potential to be used as live vaccines. We have used transposon-directed insertion site sequencing (TraDIS) to screen mutants of Salmonella enterica serovar Typhimurium for their ability to infect and grow in the tissues of wild-type and immunodeficient mice. This was to identify bacterial genes that might be deleted for the development of live attenuated vaccines that would be safer to use in situations and/or geographical areas where immunodeficiencies are prevalent. The relative fitness of each of 9,356 transposon mutants, representing mutations in 3,139 different genes, was determined in gp91 phox mice. Mutations in certain genes led to reduced fitness in both wild-type and mutant mice. To validate these results, these genes were mutated by allelic replacement, and resultant mutants were retested for fitness in the mice. A defined deletion mutant of cysE was attenuated in C57BL/6 wild-type mice and immunodeficient

[1]  A. Hill,et al.  Genetic susceptibility to invasive Salmonella disease , 2015, Nature Reviews Immunology.

[2]  J. Crump,et al.  Global Burden of Invasive Nontyphoidal Salmonella Disease, 2010 , 2015, Emerging infectious diseases.

[3]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[4]  Julian Parkhill,et al.  Comprehensive Assignment of Roles for Salmonella Typhimurium Genes in Intestinal Colonization of Food-Producing Animals , 2013, PLoS genetics.

[5]  Heng Li.  Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM , 2013, 1303.3997.

[6]  Bernadette A. Thomas,et al.  Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010 , 2012, The Lancet.

[7]  Marcel Martin.  Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[8]  J. Crump,et al.  Global trends in typhoid and paratyphoid Fever. , 2010, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[9]  Leopold Parts,et al.  Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. , 2009, Genome research.

[10]  Dov J. Stekel,et al.  Comprehensive Identification of Salmonella enterica Serovar Typhimurium Genes Required for Infection of BALB/c Mice , 2009, PLoS pathogens.

[11]  M. Gordon.  Salmonella infections in immunocompromised adults. , 2008, The Journal of infection.

[12]  Olivier Restif,et al.  Modelling within-Host Spatiotemporal Dynamics of Invasive Bacterial Disease , 2008, PLoS biology.

[13]  M. Molyneux,et al.  Epidemics of invasive Salmonella enterica serovar enteritidis and S. enterica Serovar typhimurium infection associated with multidrug resistance among adults and children in Malawi. , 2008, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[14]  I. Charles,et al.  Single, double and triple mutants of Salmonella enterica serovar Typhimurium degP (htrA), degQ (hhoA) and degS (hhoB) have diverse phenotypes on exposure to elevated temperature and their growth in vivo is attenuated to different extents. , 2006, Microbial pathogenesis.

[15]  I. Hautefort,et al.  Single-Copy Green Fluorescent Protein Gene Fusions Allow Accurate Measurement of Salmonella Gene Expression In Vitro and during Infection of Mammalian Cells , 2003, Applied and Environmental Microbiology.

[16]  C. MacLennan,et al.  Resistance and susceptibility to Salmonella infections: lessons from mice and patients with immunodeficiencies , 2003 .

[17]  G. Dougan,et al.  Salmonella typhi and S typhimurium derivatives harbouring deletions in aromatic biosynthesis and Salmonella Pathogenicity Island-2 (SPI-2) genes as vaccines and vectors. , 2003, Vaccine.

[18]  M. Molyneux,et al.  Non-typhoidal salmonella bacteraemia among HIV-infected Malawian adults: high mortality and frequent recrudescence , 2002, AIDS.

[19]  Marjan,et al.  of Healthy Volunteers ) Mutations by ssaV Secretion System ( Pathogenicity Island 2 Type III Salmonella and aroC Derivatives Harboring Defined Salmonella enterica Characterization of , 2002 .

[20]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[21]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[22]  D. Court,et al.  An efficient recombination system for chromosome engineering in Escherichia coli. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[23]  F. Fang,et al.  Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase. , 2000, Science.

[24]  A. Stewart,et al.  Rapid modification of bacterial artificial chromosomes by ET-recombination. , 1999, Nucleic acids research.

[25]  S. Clare,et al.  Interleukin-12 Is Required for Control of the Growth of Attenuated Aromatic-Compound-Dependent Salmonellae in BALB/c Mice: Role of Gamma Interferon and Macrophage Activation , 1998, Infection and Immunity.

[26]  P. Mastroeni,et al.  Correlates of protection induced by live Aro−Salmonella typhimurium vaccines in the murine typhoid model , 1997, Immunology.

[27]  P. Mastroeni,et al.  Salmonella typhimurium aroA, htrA, and aroD htrA mutants cause progressive infections in athymic (nu/nu) BALB/c mice , 1997, Infection and immunity.

[28]  G. Dougan,et al.  Safety of live oral Salmonella typhi vaccine strains with deletions in htrA and aroC aroD and immune response in humans , 1997, Infection and immunity.

[29]  G. Dougan,et al.  Construction and characterization of vaccine strains of Salmonella harboring mutations in two different aro genes. , 1988, The Journal of infectious diseases.

[30]  W. J. Dower,et al.  High efficiency transformation of E. coli by high voltage electroporation , 1988, Nucleic Acids Res..

[31]  B. Stocker,et al.  Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines , 1981, Nature.

[32]  A. C. Chang,et al.  Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid , 1978, Journal of bacteriology.

[33]  S. Falkow,et al.  Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. , 1977, Gene.

[34]  H. Northen,et al.  Deletion of tolA in Salmonella Typhimurium generates an attenuated strain with vaccine potential. , 2009, Microbiology.

[35]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .