James C. Smoot

Genome sequence of a serotype M3 strain of group A Streptococcus: Phage-encoded toxins, the high-virulence phenotype, and clone emergence

Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group A Streptococcus (GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares ≈1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A2 (designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS.

Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks

Acute rheumatic fever (ARF), a sequelae of group A Streptococcus (GAS) infection, is the most common cause of preventable childhood heart disease worldwide. The molecular basis of ARF and the subsequent rheumatic heart disease are poorly understood. Serotype M18 GAS strains have been associated for decades with ARF outbreaks in the U.S. As a first step toward gaining new insight into ARF pathogenesis, we sequenced the genome of strain MGAS8232, a serotype M18 organism isolated from a patient with ARF. The genome is a circular chromosome of 1,895,017 bp, and it shares 1.7 Mb of closely related genetic material with strain SF370 (a sequenced serotype M1 strain). Strain MGAS8232 has 178 ORFs absent in SF370. Phages, phage-like elements, and insertion sequences are the major sources of variation between the genomes. The genomes of strain MGAS8232 and SF370 encode many of the same proven or putative virulence factors. Importantly, strain MGAS8232 has genes encoding many additional secreted proteins involved in human–GAS interactions, including streptococcal pyrogenic exotoxin A (scarlet fever toxin) and two uncharacterized pyrogenic exotoxin homologues, all phage-associated. DNA microarray analysis of 36 serotype M18 strains from diverse localities showed that most regions of variation were phages or phage-like elements. Two epidemics of ARF occurring 12 years apart in Salt Lake City, UT, were caused by serotype M18 strains that were genetically identical, or nearly so. Our analysis provides a critical foundation for accelerated research into ARF pathogenesis and a molecular framework to study the plasticity of GAS genomes.

Global differential gene expression in response to growth temperature alteration in group A Streptococcus

Pathogens are exposed to different temperatures during an infection cycle and must regulate gene expression accordingly. However, the extent to which virulent bacteria alter gene expression in response to temperatures encountered in the host is unknown. Group A Streptococcus (GAS) is a human-specific pathogen that is responsible for illnesses ranging from superficial skin infections and pharyngitis to severe invasive infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS survives and multiplies at different temperatures during human infection. DNA microarray analysis was used to investigate the influence of temperature on global gene expression in a serotype M1 strain grown to exponential phase at 29°C and 37°C. Approximately 9% of genes were differentially expressed by at least 1.5-fold at 29°C relative to 37°C, including genes encoding transporter proteins, proteins involved in iron homeostasis, transcriptional regulators, phage-associated proteins, and proteins with no known homologue. Relatively few known virulence genes were differentially expressed at this threshold. However, transcription of 28 genes encoding proteins with predicted secretion signal sequences was altered, indicating that growth temperature substantially influences the extracellular proteome. TaqMan real-time reverse transcription–PCR assays confirmed the microarray data. We also discovered that transcription of genes encoding hemolysins, and proteins with inferred roles in iron regulation, transport, and homeostasis, was influenced by growth at 40°C. Thus, GAS profoundly alters gene expression in response to temperature. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many unforeseen lines of pathogenesis investigation.

Optimization of Single-Base-Pair Mismatch Discrimination in Oligonucleotide Microarrays

ABSTRACT The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.

Identification of Rgg-Regulated Exoproteins of Streptococcus pyogenes

ABSTRACT Streptococcus pyogenes secretes many proteins that influence host-pathogen interactions. Despite their importance, relatively little is known about the regulation of these proteins. Thergg gene (also known as ropB) is required for the expression of streptococcal erythrogenic toxin B (SPE B), an extracellular cysteine protease that contributes to virulence. Proteomics was used to determine if rgg regulates the expression of additional exoproteins. Exponential- and stationary-phase culture supernatant proteins made by S. pyogenes NZ131rgg and NZ131 speB were separated by two-dimensional electrophoresis. Differences were identified in supernatant proteins from both exponential- and stationary-phase cultures, although considerably more differences were detected among stationary-phase supernatant proteins. Forty-two proteins were identified by peptide fingerprinting with matrix-assisted laser desorption mass spectrometry. Mitogenic factor, DNA entry nuclease (open reading frame [ORF 226]), and ORF 953, which has no known function, were more abundant in the culture supernatants of thergg mutant compared to the speB mutant. ClpB, lysozyme, and autolysin were detected in the culture supernatant of thespeB mutant but not the rgg mutant. To determine if Rgg affected protein expression at the transcriptional level, real-time (TaqMan) reverse transcription (RT)-PCR was used to quantitate Rgg-regulated transcripts from NZ131 wild-type andspeB and rgg mutant strains. The results obtained with RT-PCR correlated with the proteomic data. We conclude that Rgg regulates the transcription of several genes expressed primarily during the stationary phase of growth.

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.

Molecular Analysis of Group A Streptococcus Type emm18 Isolates Temporally Associated with Acute Rheumatic Fever Outbreaks in Salt Lake City, Utah

ABSTRACT Acute rheumatic fever (ARF) and subsequent rheumatic heart disease are rare but serious sequelae of group A Streptococcus (GAS) infections in most western countries. Salt Lake City (SLC), Utah, and the surrounding intermountain region experienced a resurgence of ARF in 1985 which has persisted. The largest numbers of cases were encountered in 1985-1986 and in 1997-1998. Organisms with a mucoid colony phenotype when grown on blood agar plates were temporally associated with the higher incidence of ARF. To develop an understanding of the molecular population genetic structure of GAS strains associated with ARF in the SLC region, 964 mucoid and nonmucoid pharyngeal isolates recovered in SLC from 1984 to 1999 were studied by sequencing the emm gene. Isolates with an emm18 allele were further characterized by sequencing the spa, covR, and covS genes. Peak periods of ARF were associated with GAS isolates possessing an emm18 allele encoding the protein found in serotype M18 isolates. Among the serotype M18 isolates, the difference in the number of C repeats produced three size variants. Variation was limited in spa, a gene that encodes a streptococcal protective antigen, and covR and covS, genes that encode a two-component regulatory system that, when inactivated, results in a mucoid phenotype and enhanced virulence in mouse infection models. Pulsed-field gel electrophoresis showed a single restriction profile for serotype M18 organisms isolated during both peak periods of ARF. In SLC, the incidence of ARF coresurged with the occurrence of GAS serotype M18 isolates that have very restricted genetic variation.

Trophic position and potential food sources of 2 species of unionid bivalves (Mollusca:Unionidae) in 2 small Ohio streams

Abstract We determined the trophic positions of 2 species of freshwater mussels, Elliptio dilatata and Ptychobranchus fasciolaris, from 2 small streams in central Ohio by measuring stable C and N isotope ratios and digestive fluid enzyme activities. We also examined stable C and N isotopes, microbial biomass, microbial community structure, nutrient (i.e., C, N, and P) concentrations, and contribution of microbial C to total fine particulate organic C (FPOC). We hypothesized that 1) allochthonous inputs compose most of FPOC, 2) mussels use fine particulate organic material (FPOM) as a food source, and 3) mussels respond to the low-protein content of FPOM by showing high protease activity. Microbial C composed 35 to 86% of total FPOC during the autumn sampling period. FPOM stable isotope values varied seasonally, whereas δ13C and δ15N content in mussel tissue was spatially (i.e., among sites) and temporally similar. Mussels were 2 to 4‰ more depleted in δ13C than seasonal FPOM. Digestive fluid enzymes were spatially and temporally stable across species, with activity of esterase > protease > lipase > glucosidase. Lipase:protease of digestive fluids from mussels were <1, indicating a low-protein diet. Our results suggest that microbial biomass in FPOM constitutes a large portion of mussel diet and that mussels assimilate significant amounts of C from this source.

DNA microarrays as salivary diagnostic tools for characterizing the oral cavity's microbial community.

The interest in using saliva as a diagnostic medium has increased during the last decade, and recent technological developments are responsible for the advancement of its use as a diagnostic fluid (Streckfus and Bigler, 2002). There are several advantages to using saliva as a diagnostic fluid. Saliva is easy to collect, store, and ship, and, compared with the collection of blood, saliva collection is inexpensive and non-invasive, which is much safer for healthcare workers (Slavkin, 1998). In the near future, salivary diagnostic devices based on highly parallel data collection methods (e.g., DNA microarrays) will be very useful tools for health-care professionals. DNA microarrays are now used as tools for developing a comprehensive characterization of oral diseases. For example, Li et al. (2004) used high-density oligonucleotide microarrays to profile transcripts found in saliva from head and neck cancer patients, and found that thousands of human mRNAs are present in cell-free saliva. In conjunction with collaborators, our laboratory is using DNA microarrays to detect micro-organisms from the human oral cavity and, ultimately, to develop a microarray-based device for clinical applications.

Saliva-based diagnostics using 16S rRNA microarrays and microfluidics.

Abstract:  The development of a diagnostic system based on DNA microarrays for rapid identification and enumeration of microbial species in the oral cavity is described. This system uses gel‐based microarrays with immobilized probes designed within a phylogenetic framework that provides for comprehensive microbial monitoring. Understanding the community structure in the oral cavity is a necessary foundation on which to understand the breadth and depth of different microbial communities in the oral cavity and their role in acute and systemic disease. Our ultimate goal is to develop a diagnostic device to identify individuals at high risk for oral disease, and thereby reduce its prevalence and therefore the economic burden associated with treatment. This article discusses recent improvements of our system in reducing diffusional constraints in order to provide more rapid and accurate measurements of the microbial composition of saliva.