Genetic diversity of Mycobacterium tuberculosis isolates from foreign-born and Japan-born residents in Tokyo

AbstractSequences of the full genomes of 259 clinical isolates of Mycobacterium tuberculosis, obtained from foreign-born and Japan-born patients in Tokyo, Japan, were determined, and a phylogenetic tree constructed by concatenated single-nucleotide polymorphism (SNP) sequences. The 259 isolates were clustered into four clades: Lineage 2 (East Asian or “Beijing” genotype; n = 182, 70.3%), Lineage 4 (Euro-American, n = 46, 17.8%), Lineage 1 (Indo-Oceanic, n = 23, 8.9%), and Lineage 3 (East African-Indian, n = 8, 3.1%). Of the 259, 36 (13.9%) were resistant to at least one drug. There was no multi-drug-resistant isolate. Drug resistance was greater for the strains in Lineage 2 than the non-Lineage 2. The proportion of Lineage 2 isolates was significantly smaller in foreign-born (n = 43/91, 47.3%) than in Japan-born (n = 139/168, 82.7%) patients, whereas the proportion of Lineage 1 isolates was significantly larger in foreign-born (n = 19/91, 20.9%) than in Japan-born (n = 4/168, 2.4%) patients. We also found eight SNPs specific to the typical Beijing sub-genotype in Lineage 2, including 4 non-synonymous SNPs. Of the 259 isolates, 244 had strain-specific SNP(s) and small (1–30-bp) insertions and deletions (indels). The numbers of strain-specific SNPs and indels per isolate were significantly larger from foreign-born (median 89, range 0–520) than from Japan-born (median 23, range 0–415) (p 3.66E-15) patients. These results suggested that M. tuberculosis isolates from foreign-born patients had more genetic diversity than those from Japan-born patients

Acquisition of the Sda1-encoding bacteriophage does not enhance virulence of the serotype M1 Streptococcus pyogenes strain SF370

The resurgence of invasive disease caused by Streptococcus pyogenes (group A Streptococcus [GAS]) in the past 30 years has paralleled the emergence and global dissemination of the highly virulent M1T1 clone. The GAS M1T1 clone has diverged from the ancestral M1 serotype by horizontal acquisition of two unique bacteriophages, encoding the potent DNase Sda1/SdaD2 and the superantigen SpeA, respectively. The phage-encoded DNase promotes escape from neutrophil extracellular traps and is linked to enhanced virulence of the M1T1 clone. In this study, we successfully used in vitro lysogenic conversion to transfer the Sda1-encoding phage from the M1T1 clonal strain 5448 to the nonclonal M1 isolate SF370 and determined the impact of this horizontal gene transfer event on virulence. Although Sda1 was expressed in SF370 lysogens, no capacity of the phage-converted strain to survive human neutrophil killing, switch to a hyperinvasive covRS mutant form, or cause invasive lethal infection in a humanized plasminogen mouse model was observed. This work suggests that the hypervirulence of the M1T1 clone is due to the unique synergic effect of the M1T1 clone bacteriophage-specific virulence factor Sda1 acting in concert with the M1T1 clone-specific genetic scaffold

Highly Frequent Mutations in Negative Regulators of Multiple Virulence Genes in Group A Streptococcal Toxic Shock Syndrome Isolates

Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates) and non-invasive infections (59 isolates), 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%). The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors

Incompetence of Neutrophils to Invasive Group A streptococcus Is Attributed to Induction of Plural Virulence Factors by Dysfunction of a Regulator

Group A streptococcus (GAS) causes variety of diseases ranging from common pharyngitis to life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. The characteristic of invasive GAS infections has been thought to attribute to genetic changes in bacteria, however, no clear evidence has shown due to lack of an intriguingly study using serotype-matched isolates from clinical severe invasive GAS infections. In addition, rare outbreaks of invasive infections and their distinctive pathology in which infectious foci without neutrophil infiltration hypothesized us invasive GAS could evade host defense, especially neutrophil functions. Herein we report that a panel of serotype-matched GAS, which were clinically isolated from severe invasive but not from non-invaive infections, could abrogate functions of human polymorphnuclear neutrophils (PMN) in at least two independent ways; due to inducing necrosis to PMN by enhanced production of a pore-forming toxin streptolysin O (SLO) and due to impairment of PMN migration via digesting interleukin-8, a PMN attracting chemokine, by increased production of a serine protease ScpC. Expression of genes was upregulated by a loss of repressive function with the mutation of csrS gene in the all emm49 severe invasive GAS isolates. The csrS mutants from clinical severe invasive GAS isolates exhibited high mortality and disseminated infection with paucity of neutrophils, a characteristic pathology seen in human invasive GAS infection, in a mouse model. However, GAS which lack either SLO or ScpC exhibit much less mortality than the csrS-mutated parent invasive GAS isolate to the infected mice. These results suggest that the abilities of GAS to abrogate PMN functions can determine the onset and severity of invasive GAS infection

CovR-Controlled Global Regulation of Gene Expression in Streptococcus mutans

CovR/S is a two-component signal transduction system (TCS) that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (∼6.5% of the genome) were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI). Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus

Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (ΔsseMGAS5005) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. KM and kcat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of ΔsseMGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses

Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique

We thank the members of the Laboratory of Microbial Pathogenesis and Immunology, especially Annette Nelkenbaum and Ben Winer for their technical assistance. We also thank Estee Colleen Cervantes and Sutapa Banerjee from Hunter College for their technical contribution to this project. We are grateful to Joseph Ferretti for S. pyogenes strain SF370.Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.Yeshttp://www.plosone.org/static/editorial#pee