Nancy P. Hoe

Evolutionary Origin and Emergence of a Highly Successful Clone of Serotype M1 Group A Streptococcus Involved Multiple Horizontal Gene Transfer Events

To better understand the molecular events involved in the origin of new pathogenic bacteria, we studied the evolution of a highly virulent clone of serotype M1 group A Streptococcus (GAS). Genomic, DNA-DNA microarray, and single-nucleotide polymorphism analyses indicated that this clone evolved through a series of horizontal gene transfer events that involved (1) the acquisition of prophages encoding streptococcal pyrogenic exotoxin A and extracellular DNases and (2) the reciprocal recombination of a 36-kb chromosomal region encoding the extracellular toxins NAD+-glycohydrolase (NADase) and streptolysin O (SLO). These gene transfer events were associated with significantly increased production of SLO and NADase. Virtual identity in the 36-kb region present in contemporary serotype M1 and M12 isolates suggests that a serotype M12 strain served as the donor of this region. Multiple horizontal gene transfer events were a crucial factor in the evolutionary origin and emergence of a very abundant contemporary clone of serotype M1 GAS.

Evasion of human innate and acquired immunity by a bacterial homolog of CD11b that inhibits opsonophagocytosis

Microbial pathogens must evade the human immune system to survive, disseminate and cause disease. By proteome analysis of the bacterium Group A Streptococcus (GAS), we identified a secreted protein with homology to the α-subunit of Mac-1, a leukocyte β2 integrin required for innate immunity to invading microbes. The GAS Mac-1–like protein (Mac) was secreted by most pathogenic strains, produced in log-phase and controlled by the covR-covS two-component gene regulatory system, which also regulates transcription of other GAS virulence factors. Patients with GAS infection had titers of antibody specific to Mac that correlated with the course of disease, demonstrating that Mac was produced in vivo. Mac bound to CD16 (FcγRIIIB) on the surface of human polymorphonuclear leukocytes and inhibited opsonophagocytosis and production of reactive oxygen species, which resulted in significantly decreased pathogen killing. Thus, by mimicking a host-cell receptor required for an innate immune response, the GAS Mac protein inhibits professional phagocyte function by a novel strategy that enhances pathogen survival, establishment of infection and dissemination.

Nonpolar Inactivation of the Hypervariable Streptococcal Inhibitor of Complement Gene (sic) in Serotype M1 Streptococcus pyogenes Significantly Decreases Mouse Mucosal Colonization

ABSTRACT Group A Streptococcus (GAS) is a human pathogen that commonly infects the upper respiratory tract. GAS serotype M1 strains are frequently isolated from human infections and contain the gene encoding the hypervariable streptococcal inhibitor of complement protein (Sic). It was recently shown that Sic variants were rapidly selected on mucosal surfaces in epidemic waves caused by M1 strains, an observation suggesting that Sic participates in host-pathogen interactions on the mucosal surface (N. P. Hoe, K. Nakashima, S. Lukomski, D. Grigsby, M. Liu, P. Kordari, S.-J. Dou, X. Pan, J. Vuopio-Varkila, S. Salmelinna, A. McGeer, D. E. Low, B. Schwartz, A. Schuchat, S. Naidich, D. De Lorenzo, Y.-X. Fu, and J. M. Musser, Nat. Med. 5:924–929, 1999). To test this idea, a new nonpolar mutagenesis method employing a spectinomycin resistance cassette was used to inactivate the sic gene in an M1 GAS strain. The isogenic Sic-negative mutant strain was significantly (P < 0.019) impaired in ability to colonize the mouse mucosal surface after intranasal infection. These results support the hypothesis that the predominance of M1 strains in human infections is related, in part, to a Sic-mediated enhanced colonization ability.

Genome Sequence of a Lancefield Group C Streptococcus zooepidemicus Strain Causing Epidemic Nephritis: New Information about an Old Disease

Outbreaks of disease attributable to human error or natural causes can provide unique opportunities to gain new information about host-pathogen interactions and new leads for pathogenesis research. Poststreptococcal glomerulonephritis (PSGN), a sequela of infection with pathogenic streptococci, is a common cause of preventable kidney disease worldwide. Although PSGN usually occurs after infection with group A streptococci, organisms of Lancefield group C and G also can be responsible. Despite decades of study, the molecular pathogenesis of PSGN is poorly understood. As a first step toward gaining new information about PSGN pathogenesis, we sequenced the genome of Streptococcus equi subsp. zooepidemicus strain MGCS10565, a group C organism that caused a very large and unusually severe epidemic of nephritis in Brazil. The genome is a circular chromosome of 2,024,171 bp. The genome shares extensive gene content, including many virulence factors, with genetically related group A streptococci, but unexpectedly lacks prophages. The genome contains many apparently foreign genes interspersed around the chromosome, consistent with the presence of a full array of genes required for natural competence. An inordinately large family of genes encodes secreted extracellular collagen-like proteins with multiple integrin-binding motifs. The absence of a gene related to speB rules out the long-held belief that streptococcal pyrogenic exotoxin B or antibodies reacting with it singularly cause PSGN. Many proteins previously implicated in GAS PSGN, such as streptokinase, are either highly divergent in strain MGCS10565 or are not more closely related between these species than to orthologs present in other streptococci that do not commonly cause PSGN. Our analysis provides a comparative genomics framework for renewed appraisal of molecular events underlying APSGN pathogenesis.

Insight into the molecular basis of pathogen abundance: Group A Streptococcus inhibitor of complement inhibits bacterial adherence and internalization into human cells

Streptococcal inhibitor of complement (Sic) is a secreted protein made predominantly by serotype M1 Group A Streptococcus (GAS), which contributes to persistence in the mammalian upper respiratory tract and epidemics of human disease. Unexpectedly, an isogenic sic-negative mutant adhered to human epithelial cells significantly better than the wild-type parental strain. Purified Sic inhibited the adherence of a sic negative serotype M1 mutant and of non-Sic-producing GAS strains to human epithelial cells. Sic was rapidly internalized by human epithelial cells, inducing cell flattening and loss of microvilli. Ezrin and moesin, human proteins that functionally link the cytoskeleton to the plasma membrane, were identified as Sic-binding proteins by affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Sic colocalized with ezrin inside epithelial cells and bound to the F-actin-binding site region located in the carboxyl terminus of ezrin and moesin. Synthetic peptides corresponding to two regions of Sic had GAS adherence-inhibitory activity equivalent to mature Sic and inhibited binding of Sic to ezrin. In addition, the sic mutant was phagocytosed and killed by human polymorphonuclear leukocytes significantly better than the wild-type strain, and Sic colocalized with ezrin in discrete regions of polymorphonuclear leukocytes. The data suggest that binding of Sic to ezrin alters cellular processes critical for efficient GAS contact, internalization, and killing. Sic enhances bacterial survival by enabling the pathogen to avoid the intracellular environment. This process contributes to the abundance of M1 GAS in human infections and their ability to cause epidemics.

Characterization of two novel pyrogenic toxin superantigens made by an acute rheumatic fever clone of Streptococcus pyogenes associated with multiple disease outbreaks.

ABSTRACT The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designated speL and speM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS). speL and speM were located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS. speL and speM were transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vβ1, Vβ5.1, and Vβ23, and SpeM had specificity for Vβ1 and Vβ23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.

Group A Streptococcus Gene Expression in Humans and Cynomolgus Macaques with Acute Pharyngitis

ABSTRACT The molecular mechanisms used by group A Streptococcus (GAS) to survive on the host mucosal surface and cause acute pharyngitis are poorly understood. To provide new information about GAS host-pathogen interactions, we used real-time reverse transcription-PCR (RT-PCR) to analyze transcripts of 17 GAS genes in throat swab specimens taken from 18 pediatric patients with pharyngitis. The expression of known and putative virulence genes and regulatory genes (including genes in seven two-component regulatory systems) was studied. Several known and previously uncharacterized GAS virulence gene regulators were highly expressed compared to the constitutively expressed control gene proS. To examine in vivo gene transcription in a controlled setting, three cynomolgus macaques were infected with strain MGAS5005, an organism that is genetically representative of most serotype M1 strains recovered from pharyngitis and invasive disease episodes in North America and Western Europe. These three animals developed clinical signs and symptoms of GAS pharyngitis and seroconverted to several GAS extracellular proteins. Real-time RT-PCR analysis of throat swab material collected at intervals throughout a 12-day infection protocol indicated that expression profiles of a subset of GAS genes accurately reflected the profiles observed in the human pediatric patients. The results of our study demonstrate that analysis of in vivo GAS gene expression is feasible in throat swab specimens obtained from infected human and nonhuman primates. In addition, we conclude that the cynomolgus macaque is a useful nonhuman primate model for the study of molecular events contributing to acute pharyngitis caused by GAS.

M Types of Group A Streptococcal Isolates Submitted to the National Centre for Streptococcus (Canada) from 1993 to 1999

ABSTRACT The National Centre for Streptococcus (NCS) (Canada) determined the group A streptococcal (GAS) M types of 4,760 Canadian isolates submitted between 1993 and 1999 by classic serotyping. The 10 most frequently identified M types were M1 (26.4%), M12 (9.8%), M28 (8.9%), M3 (6.8%), M4 (6.2%), M11 (4.8%), M89 (3.1%), M6 (3.0%), M2 (2.6%), and M77 (1.9%). Nontypeable isolates accounted for 15.4% of the collection. The province of Ontario submitted 51.1% of the isolates, followed by Quebec (21.2%) and Alberta (13.9%). Together, these three provinces constituted 71.3% of the Canadian population in 1996. The numbers of M types M1, M12, M28, and M3 occurred most frequently in subjects whose ages were <1 to 15 years and 25 to 45 years, as well as in the elderly (60 to 90 years). Further analysis found that the four most frequently identified M types from blood, brain, and cerebrospinal fluid were M1 (28.2%), M28 (9.2%), M12 (9.1%), and M3 (8.2%), with 13.4% of isolates being nontypeable. The four isolates from throats most frequently identified were M1 (19.5%), M12 (15.3%), M3 (8.6%), and M28 (5%) with 19.4% of isolates being nontypeable. The sic gene of a subset of M1 strains (9.5% of the M1 collection) was sequenced. Of 36 sic types identified, the four most common were sic1.01 (22.8%), sic1.02 (14.9%), sic1.135 (10.5%), and sic1.178 (9.6%). Together these four sic types further characterized nearly 60% of the M1 strains sequenced. In summary, from the years 1993 to 1999, the NCS detected 54 M types, of which 10 different M types constituted 73.5% of the collection. M1 was the most common GAS M type circulating in the Canadian population, responsible for more than a quarter of the isolates typed. The most common throat isolates differed in M-type and proportion from those of invasive isolates. Sequencing the sic gene further characterized the most common M-type serotype 1 in a fashion that may be useful for epidemiologic investigations.

The Heterogeneity of Endemic Community Pediatric Group A Streptococcal Pharyngeal Isolates and Their Relationship to Invasive Isolates

By use of molecular techniques, the genetic heterogeneity of 63 community pediatric pharyngeal group A streptococcal (GAS) isolates circulating within a 3-week period were compared with 17 contemporaneous invasive pediatric isolates. Pharyngitis isolates represented 16 pulsed-field gel electrophoresis (PFGE) patterns with 12 emm serotypes, and invasive isolates represented 10 PFGE patterns with 9 emm serotypes. One-fourth of the pharyngeal isolates (16/63) were identical to at least 1 invasive isolate; conversely, 10 (59%) of 17 invasive isolates were identical to at least 1 pharyngeal strain. sic allele analysis of emm1 strains demonstrated additional heterogeneity and overlap. More pharyngeal (71%) than invasive isolates (35%) were positive for both speA and speC (P<.02). Many pharyngitis GAS strains circulate simultaneously. Most invasive pediatric GAS strains are identical to acute pharyngitis strains; thus, childhood pharyngitis is a major reservoir for strains with invasive potential. Pharyngeal isolates were more likely to be speA and speC positive than were the invasive isolates.

Distribution of streptococcal inhibitor of complement variants in pharyngitis and invasive isolates in an epidemic of serotype M1 group A Streptococcus infection.

Streptococcal inhibitor of complement (Sic) is a highly polymorphic extracellular protein made predominantly by serotype M1 group A Streptococcus (GAS). New variants of the Sic protein frequently appear in M1 epidemics as a result of positive natural selection. To gain further understanding of the molecular basis of M1 epidemics, the sic gene was sequenced from 471 pharyngitis and 127 pyogenic and blood isolates recovered from 598 patients living in metropolitan Helsinki, Finland, during a 37-month population-based surveillance study. Most M1 GAS subclones recovered from pyogenic infections and blood were abundantly represented in the pool of subclones causing pharyngitis. Alleles shared among the pharyngitis, pyogenic, and blood samples were identified in throat isolates a mean of 9.8 months before their recovery from pyogenic infections and blood, which indicates that selection of most sic variants occurs on mucosal surfaces. In contrast, no variation was identified in the emm and covR/covS genes.