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1.  Molecular Characterization of an Invasive Phenotype of Group A Streptococcus Arising During Human Infection Using Whole Genome Sequencing of Multiple Isolates From the Same Patient 
The Journal of Infectious Diseases  2013;209(10):1520-1523.
Invasive group A streptococcal (GAS) strains often have genetic differences compared to GAS strains from nonsterile sites. Invasive, “hypervirulent” GAS strains can arise from a noninvasive progenitor following subcutaneous inoculation in mice, but such emergence has been rarely characterized in humans. We used whole genome analyses of multiple GAS isolates from the same patient to document the molecular basis for emergence of a GAS strain with an invasive phenotype during human infection. In contrast to previous theories, we found that elimination of production of the cysteine protease SpeB was not necessary for emergence of GAS with an invasive, “hypervirulent” phenotype.
PMCID: PMC3997578  PMID: 24307742
group A Streptococcus; bacterial pathogenesis; virulence; whole genome sequencing; gene expression
2.  Asymptomatic Carriage of Group A Streptococcus Is Associated with Elimination of Capsule Production 
Infection and Immunity  2014;82(9):3958-3967.
Humans commonly carry pathogenic bacteria asymptomatically, but despite decades of study, the underlying molecular contributors remain poorly understood. Here, we show that a group A streptococcus carriage strain contains a frameshift mutation in the hasA gene resulting in loss of hyaluronic acid capsule biosynthesis. This mutation was repaired by allelic replacement, resulting in restoration of capsule production in the isogenic derivative strain. The “repaired” isogenic strain was significantly more virulent than the carriage strain in a mouse model of necrotizing fasciitis and had enhanced growth ex vivo in human blood. Importantly, the repaired isogenic strain colonized the mouse oropharynx with significantly greater bacterial burden and had significantly reduced ability to internalize into cultured epithelial cells than the acapsular carriage strain. We conducted full-genome sequencing of 81 strains cultured serially from 19 epidemiologically unrelated human subjects and discovered the common theme that mutations negatively affecting capsule biosynthesis arise in vivo in the has operon. The significantly decreased capsule production is a key factor contributing to the molecular détente between pathogen and host. Our discoveries suggest a general model for bacterial pathogens in which mutations that downregulate or ablate virulence factor production contribute to carriage.
PMCID: PMC4187823  PMID: 25024363
3.  GyrB Polymorphisms Accurately Assign Invasive Viridans Group Streptococcal Species 
Journal of Clinical Microbiology  2014;52(8):2905-2912.
Viridans group streptococci (VGS) are a heterogeneous group of medically important bacteria that cannot be accurately assigned to a particular species using conventional phenotypic methods. Although multilocus sequence analysis (MLSA) is considered the gold standard for VGS species-level identification, MLSA is not yet feasible in the clinical setting. Conversely, molecular methods, such as sodA and 16S rRNA gene sequencing, are clinically practical but not sufficiently accurate for VGS species-level identification. Here, we present data regarding the use of an ∼400-nucleotide internal fragment of the gene encoding DNA gyrase subunit B (GyrB) for VGS species-level identification. MLSA, internal gyrB, sodA, full-length, and 5′ 16S gene sequences were used to characterize 102 unique VGS blood isolates collected from 2011 to 2012. When using the MLSA species assignment as a reference, full-length and 5′ partial 16S gene and sodA sequence analyses failed to correctly assign all strains to a species. Precise species determination was particularly problematic for Streptococcus mitis and Streptococcus oralis isolates. However, the internal gyrB fragment allowed for accurate species designations for all 102 strains. We validated these findings using 54 VGS strains for which MLSA, 16S gene, sodA, and gyrB data are available at the NCBI, showing that gyrB is superior to 16S gene and sodA sequence analyses for VGS species identification. We also observed that specific polymorphisms in the 133-amino acid sequence of the internal GyrB fragment can be used to identify invasive VGS species. Thus, the GyrB amino acid sequence may offer a more practical and accurate method for classifying invasive VGS strains to the species level.
PMCID: PMC4136163  PMID: 24899021
4.  Natural Variation in the Promoter of the Gene Encoding the Mga Regulator Alters Host-Pathogen Interactions in Group A Streptococcus Carrier Strains 
Infection and Immunity  2013;81(11):4128-4138.
Humans commonly carry pathogenic bacteria asymptomatically, but the molecular factors underlying microbial asymptomatic carriage are poorly understood. We previously reported that two epidemiologically unassociated serotype M3 group A Streptococcus (GAS) carrier strains had an identical 12-bp deletion in the promoter of the gene encoding Mga, a global positive gene regulator. Herein, we report on studies designed to test the hypothesis that the identified 12-bp deletion in the mga promoter alters GAS virulence, thereby potentially contributing to the asymptomatic carrier phenotype. Using allelic exchange, we introduced the variant promoter into a serotype M3 invasive strain and the wild-type promoter into an asymptomatic carrier strain. Compared to strains with the wild-type mga promoter, we discovered that strains containing the promoter with the 12-bp deletion produced significantly fewer mga and Mga-regulated gene transcripts. Consistent with decreased mga transcripts, strains containing the variant mga promoter were also significantly less virulent in in vivo and ex vivo models of GAS disease. Further, we provide evidence that the pleiotropic regulator protein CodY binds to the mga promoter and that the 12-bp deletion in the mga promoter reduces CodY-mediated mga transcription. We conclude that the naturally occurring 12-bp deletion in the mga promoter significantly alters the pathogen-host interaction of these asymptomatic carrier strains. Our findings provide new insight into the molecular basis of the carrier state of an important human pathogen.
PMCID: PMC3811816  PMID: 23980109
5.  Dual-Site Phosphorylation of the Control of Virulence Regulator Impacts Group A Streptococcal Global Gene Expression and Pathogenesis 
PLoS Pathogens  2014;10(5):e1004088.
Phosphorylation relays are a major mechanism by which bacteria alter transcription in response to environmental signals, but understanding of the functional consequences of bacterial response regulator phosphorylation is limited. We sought to characterize how phosphorylation of the control of virulence regulator (CovR) protein from the major human pathogen group A Streptococcus (GAS) influences GAS global gene expression and pathogenesis. CovR mainly serves to repress GAS virulence factor-encoding genes and has been shown to homodimerize following phosphorylation on aspartate-53 (D53) in vitro. We discovered that CovR is phosphorylated in vivo and that such phosphorylation is partially heat-stable, suggesting additional phosphorylation at non-aspartate residues. Using mass spectroscopy along with targeted mutagenesis, we identified threonine-65 (T65) as an additional CovR phosphorylation site under control of the serine/threonine kinase (Stk). Phosphorylation on T65, as mimicked by the recombinant CovR T65E variant, abolished in vitro CovR D53 phosphorylation. Similarly, isoallelic GAS strains that were either unable to be phosphorylated at D53 (CovR-D53A) or had functional constitutive phosphorylation at T65 (CovR-T65E) had essentially an identical gene repression profile to each other and to a CovR-inactivated strain. However, the CovR-D53A and CovR-T65E isoallelic strains retained the ability to positively influence gene expression that was abolished in the CovR-inactivated strain. Consistent with these observations, the CovR-D53A and CovR-T65E strains were hypervirulent compared to the CovR-inactivated strain in a mouse model of invasive GAS disease. Surprisingly, an isoalleic strain unable to be phosphorylated at CovR T65 (CovR-T65A) was hypervirulent compared to the wild-type strain, as auto-regulation of covR gene expression resulted in lower covR gene transcript and CovR protein levels in the CovR-T65A strain. Taken together, these data establish that CovR is phosphorylated in vivo and elucidate how the complex interplay between CovR D53 activating phosphorylation, T65 inhibiting phosphorylation, and auto-regulation impacts streptococcal host-pathogen interaction.
Author Summary
Group A Streptococcus (GAS) causes a variety of human diseases ranging from mild throat infections to deadly invasive infections. The capacity of GAS to cause infections at such diverse locations is dependent on its ability to precisely control the production of a broad variety of virulence factors. The control of virulence regulator (CovR) is a master regulator of GAS genes encoding virulence factors. It is known that CovR can be phosphorylated on aspartate-53 in vitro and that such phosphorylation increases its regulatory activity, but what additional factors influence CovR-mediated gene expression have not been established. Herein we show for the first time that CovR is phosphorylated in vivo and that phosphorylation of CovR on threonine-65 by the threonine/serine kinase Stk prevents aspartate-53 phosphorylation, thereby decreasing CovR regulatory activity. Further, while CovR-mediated gene repression is highly dependent on aspartate-53 phosphorylation, CovR-mediated gene activation proceeds via a phosphorylation-independent mechanism. Modifications in CovR phosphorylation sites significantly affected the expression of GAS virulence factors during infection and markedly altered the ability of GAS to cause disease in mice. These data establish that multiple inter-related pathways converge to influence CovR phosphorylation, thereby providing new insight into the complex regulatory network used by GAS during infection.
PMCID: PMC4006921  PMID: 24788524
6.  Streptococcus mitis Strains Causing Severe Clinical Disease in Cancer Patients 
Emerging Infectious Diseases  2014;20(5):762-771.
The genetically diverse viridans group streptococci (VGS) are increasingly recognized as the cause of a variety of human diseases. We used a recently developed multilocus sequence analysis scheme to define the species of 118 unique VGS strains causing bacteremia in patients with cancer; Streptococcus mitis (68 patients) and S. oralis (22 patients) were the most frequently identified strains. Compared with patients infected with non–S. mitis strains, patients infected with S. mitis strains were more likely to have moderate or severe clinical disease (e.g., VGS shock syndrome). Combined with the sequence data, whole-genome analyses showed that S. mitis strains may more precisely be considered as >2 species. Furthermore, we found that multiple S. mitis strains induced disease in neutropenic mice in a dose-dependent fashion. Our data define the prominent clinical effect of the group of organisms currently classified as S. mitis and lay the groundwork for increased understanding of this understudied pathogen.
PMCID: PMC4012796  PMID: 24750901
viridans group streptococci; bacteremia; neutropenia; Pitt bacteremia score; Streptococcus mitis; cancer patients; cancer; multilocus sequence analysis; bacteria; characterization
7.  Polymorphisms in Regulator of Protease B (RopB) Alter Disease Phenotype and Strain Virulence of Serotype M3 Group A Streptococcus 
The Journal of Infectious Diseases  2012;205(11):1719-1729.
Whole-genome sequencing of serotype M3 group A streptococci (GAS) from oropharyngeal and invasive infections in Ontario recently showed that the gene encoding regulator of protease B (RopB) is highly polymorphic in this population. To test the hypothesis that ropB is under diversifying selective pressure among all serotype M3 GAS strains, we sequenced this gene in 1178 strains collected from different infection types, geographic regions, and time periods. The results confirmed our hypothesis and discovered a significant association between mutant ropB alleles, decreased activity of its major regulatory target SpeB, and pharyngitis. Additionally, isoallelic strains with ropB polymorphisms were significantly less virulent in a mouse model of necrotizing fasciitis. These studies provide a model strategy for applying whole-genome sequencing followed by deep single-gene sequencing to generate new insight to the rapid evolution and virulence regulation of human pathogens.
PMCID: PMC4447876  PMID: 22262791
8.  Structural mechanism of Staphylococcus aureus Hfq binding to an RNA A-tract 
Nucleic Acids Research  2012;40(21):11023-11035.
Hfq is a post-transcriptional regulator that plays a key role in bacterial gene expression by binding AU-rich sequences and A-tracts to facilitate the annealing of sRNAs to target mRNAs and to affect RNA stability. To understand how Hfq from the Gram-positive bacterium Staphylococcus aureus (Sa) binds A-tract RNA, we determined the crystal structure of an Sa Hfq–adenine oligoribonucleotide complex. The structure reveals a bipartite RNA-binding motif on the distal face that is composed of a purine nucleotide-specificity site (R-site) and a non-discriminating linker site (L-site). The (R–L)-binding motif, which is also utilized by Bacillus subtilis Hfq to bind (AG)3A, differs from the (A–R–N) tripartite poly(A) RNA-binding motif of Escherichia coli Hfq whereby the Sa Hfq R-site strongly prefers adenosine, is more aromatic and permits deeper insertion of the adenine ring. R-site adenine-stacking residue Phe30, which is conserved among Gram-positive bacterial Hfqs, and an altered conformation about β3 and β4 eliminate the adenosine-specificity site (A-site) and create the L-site. Binding studies show that Sa Hfq binds (AU)3A ≈ (AG)3A ≥ (AC)3A > (AA)3A and L-site residue Lys33 plays a significant role. The (R–L) motif is likely utilized by Hfqs from most Gram-positive bacteria to bind alternating (A–N)n RNA.
PMCID: PMC3505971  PMID: 22965117
9.  Niche-specific contribution to streptococcal virulence of a MalR-regulated carbohydrate binding protein 
Molecular microbiology  2011;81(2):500-514.
Low G+C Gram-positive bacteria typically contain multiple LacI/GalR regulator family members which often have highly similar amino-terminal DNA binding domains suggesting significant overlap in target DNA sequences. The LacI/GalR family regulator catabolite control protein A (CcpA) is a global regulator of the Group A Streptococcus (GAS) transcriptome and contributes to GAS virulence in diverse infection sites. Herein, we studied the role of the maltose repressor (MalR), another LacI/GalR family member, in GAS global gene expression and virulence. MalR inactivation reduced GAS colonization of the mouse orophyarnx but did not detrimentally affect invasive infection. The MalR transcriptome was limited to only 25 genes, and a highly conserved MalR DNA-binding sequence was identified. Variation of the MalR binding sequence significantly reduced MalR binding in vitro. In contrast, CcpA bound to the same DNA sequences as MalR but tolerated variation in the promoter sequences with minimal change in binding affinity. Inactivation of pulA, a MalR regulated gene which encodes a cell-surface carbohydrate binding protein, significantly reduced GAS human epithelial cell adhesion and mouse oropharyngeal colonization but did not affect GAS invasive disease. These data delineate a molecular mechanism by which hierarchical regulation of carbon source utilization influences bacterial pathogenesis in a site-specific fashion.
PMCID: PMC3147245  PMID: 21645132
Streptococcus; maltodextrin; pharyngitis; adhesion
10.  Distinct Single Amino Acid Replacements in the Control of Virulence Regulator Protein Differentially Impact Streptococcal Pathogenesis 
PLoS Pathogens  2011;7(10):e1002311.
Sequencing of invasive strains of group A streptococci (GAS) has revealed a diverse array of single nucleotide polymorphisms in the gene encoding the control of virulence regulator (CovR) protein. However, there is limited information regarding the molecular mechanisms by which CovR single amino acid replacements impact GAS pathogenesis. The crystal structure of the CovR C-terminal DNA-binding domain was determined to 1.50 Å resolution and revealed a three-stranded β-sheet followed by a winged helix-turn-helix DNA binding motif. Modeling of the CovR protein-DNA complex indicated that CovR single amino acid replacements observed in clinical GAS isolates could directly alter protein-DNA interaction and impact protein structure. Isoallelic GAS strains that varied by a single amino acid replacement in the CovR DNA binding domain had significantly different transcriptomes compared to wild-type and to each other. Similarly, distinct recombinant CovR variants had differential binding affinity for DNA from the promoter regions of several virulence factor-encoding genes. Finally, mice that were challenged with GAS CovR isoallelic strains had significantly different survival times, which correlated with the transcriptome and protein-DNA binding studies. Taken together, these data provide structural and functional insights into the critical and distinct effects of variation in the CovR protein on GAS pathogenesis.
Author Summary
Group A Streptococcus (GAS) causes a variety of human infections including invasive disease that can often be deadly. GAS strains that cause serious infections may have alterations in the amino acid sequence of the control of virulence regulator (CovR) protein, but mechanisms by which changes in the CovR protein influence GAS disease are not understood. We determined the crystal structure of the CovR DNA binding region and found that alterations in the CovR protein observed in clinical, invasive GAS isolates are likely to disrupt CovR-DNA interaction and overall CovR structure. In accord with the structural data, CovR proteins with a single amino acid change had distinctly different binding affinities for various GAS virulence-factor encoding genes. Similarly, GAS strains that differed by only the presence of a single CovR amino acid change had distinct gene expression profiles. Finally, mice that were challenged with GAS strains that differed by only a single CovR amino acid replacement had significantly different survival times consistent with the idea that alterations in the CovR protein are a key determinant of clinical outcomes in GAS human infections. These findings provide mechanistic insights into how subtle genetic differences can profoundly impact the severity of bacterial infections.
PMCID: PMC3197619  PMID: 22028655
11.  Naturally occurring single amino acid replacements in a regulatory protein alter streptococcal gene expression and virulence in mice 
The Journal of Clinical Investigation  2011;121(5):1956-1968.
Infection with different strains of the same species of bacteria often results in vastly different clinical outcomes. Despite extensive investigation, the genetic basis of microbial strain-specific virulence remains poorly understood. Recent whole-genome sequencing has revealed that SNPs are the most prevalent form of genetic diversity among different strains of the same species of bacteria. For invasive serotype M3 group A streptococci (GAS) strains, the gene encoding regulator of proteinase B (RopB) has the highest frequency of SNPs. Here, we have determined that ropB polymorphisms alter RopB function and modulate GAS host-pathogen interactions. Sequencing of ropB in 171 invasive serotype M3 GAS strains identified 19 distinct ropB alleles. Inactivation of the ropB gene in strains producing distinct RopB variants had dramatically divergent effects on GAS global gene expression. Additionally, generation of isoallelic GAS strains differing only by a single amino acid in RopB confirmed that variant proteins affected transcript levels of the gene encoding streptococcal proteinase B, a major RopB-regulated virulence factor. Comparison of parental, RopB-inactivated, and RopB isoallelic strains in mouse infection models demonstrated that ropB polymorphisms influence GAS virulence and disease manifestations. These data detail a paradigm in which unbiased, whole-genome sequence analysis of populations of clinical bacterial isolates creates new avenues of productive investigation into the pathogenesis of common human infections.
PMCID: PMC3083769  PMID: 21490401
12.  The role of complex carbohydrate catabolism in the pathogenesis of invasive streptococci 
Trends in microbiology  2008;16(7):318-325.
Historically, the study of bacterial catabolism of complex carbohydrates has contributed to understanding basic bacterial physiology. Recently, however, genome-wide screens of streptococcal pathogenesis have identified genes encoding proteins involved in complex carbohydrate catabolism as participating in pathogen infectivity. Subsequent studies have focused on specific mechanisms by which carbohydrate utilization proteins might contribute to the ability of streptococci to colonize and infect the host. Moreover, transcriptome and biochemical analyses have uncovered novel regulatory pathways by which streptococci link environmental carbohydrate availability to virulence factor production. Herein we review new insights into the role of complex carbohydrates in streptococcal host-pathogen interaction.
PMCID: PMC2975494  PMID: 18508271
13.  A Combination of Independent Transcriptional Regulators Shapes Bacterial Virulence Gene Expression during Infection 
PLoS Pathogens  2010;6(3):e1000817.
Transcriptional regulatory networks are fundamental to how microbes alter gene expression in response to environmental stimuli, thereby playing a critical role in bacterial pathogenesis. However, understanding how bacterial transcriptional regulatory networks function during host-pathogen interaction is limited. Recent studies in group A Streptococcus (GAS) suggested that the transcriptional regulator catabolite control protein A (CcpA) influences many of the same genes as the control of virulence (CovRS) two-component gene regulatory system. To provide new information about the CcpA and CovRS networks, we compared the CcpA and CovR transcriptomes in a serotype M1 GAS strain. The transcript levels of several of the same genes encoding virulence factors and proteins involved in basic metabolic processes were affected in both ΔccpA and ΔcovR isogenic mutant strains. Recombinant CcpA and CovR bound with high-affinity to the promoter regions of several co-regulated genes, including those encoding proteins involved in carbohydrate and amino acid metabolism. Compared to the wild-type parental strain, ΔccpA and ΔcovRΔccpA isogenic mutant strains were significantly less virulent in a mouse myositis model. Inactivation of CcpA and CovR alone and in combination led to significant alterations in the transcript levels of several key GAS virulence factor encoding genes during infection. Importantly, the transcript level alterations in the ΔccpA and ΔcovRΔccpA isogenic mutant strains observed during infection were distinct from those occurring during growth in laboratory medium. These data provide new knowledge regarding the molecular mechanisms by which pathogenic bacteria respond to environmental signals to regulate virulence factor production and basic metabolic processes during infection.
Author Summary
Group A Streptococcus (GAS) causes diverse infections in humans ranging from pharyngitis (strep throat) to necrotizing fasciitis (the flesh-eating disease). It is well known that GAS secretes a broad array of virulence factors that are critical to its ability to cause human infections, but how GAS coordinates virulence factor production during infection is poorly understood. We discovered that two GAS proteins, catabolite control protein A (CcpA) and control of virulence regulator (CovR), regulate production of many of the same virulence factor encoding genes, indicating that GAS uses these two regulatory proteins to modulate virulence factor production in response to environmental stimuli. We determined that CcpA and CovR are able to bind to DNA from co-regulated genes, indicating that the proteins control gene expression by directly interacting with DNA. Using a mouse model of muscle infection, we found that CcpA and CovR, alone and in combination, are critical to the ability of GAS to regulate expression of virulence factor encoding genes during infection. These findings increase understanding regarding the regulatory mechanisms critical to the ability of bacterial pathogens to cause infection.
PMCID: PMC2841617  PMID: 20333240
14.  CovS Simultaneously Activates and Inhibits the CovR-Mediated Repression of Distinct Subsets of Group A Streptococcus Virulence Factor-Encoding Genes▿ †  
Infection and Immunity  2009;77(8):3141-3149.
To colonize and cause disease at distinct anatomical sites, bacterial pathogens must tailor gene expression in a microenvironment-specific manner. The molecular mechanisms that control the ability of the human bacterial pathogen group A Streptococcus (GAS) to transition between infection sites have yet to be fully elucidated. A key regulator of GAS virulence gene expression is the CovR-CovS two-component regulatory system (also known as CsrR-CsrS). covR and covS mutant strains arise spontaneously during invasive infections and, in in vivo models of infection, rapidly become dominant. Here, we compared wild-type GAS with covR, covS, and covRS isogenic mutant strains to investigate the heterogeneity in the types of natural mutations that occur in covR and covS and the phenotypic consequences of covR or covS mutation. We found that the response regulator CovR retains some regulatory function in the absence of CovS and that CovS modulates CovR to significantly enhance repression of one group of genes (e.g., the speA, hasA, and ska genes) while it reduces repression of a second group of genes (e.g., the speB, grab, and spd3 genes). We also found that different in vivo-induced covR mutations can lead to strikingly different transcriptomes. While covS mutant strains show increased virulence in several invasive models of infection, we determined that these mutants are significantly outcompeted by wild-type GAS during growth in human saliva, an ex vivo model of upper respiratory tract infection. We propose that CovS-mediated regulation of CovR activity plays an important role in the ability of GAS to cycle between pharyngeal and invasive infections.
PMCID: PMC2715666  PMID: 19451242
15.  A decade of molecular pathogenomic analysis of group A Streptococcus  
The Journal of Clinical Investigation  2009;119(9):2455-2463.
Molecular pathogenomic analysis of the human bacterial pathogen group A Streptococcus has been conducted for a decade. Much has been learned as a consequence of the confluence of low-cost DNA sequencing, microarray technology, high-throughput proteomics, and enhanced bioinformatics. These technical advances, coupled with the availability of unique bacterial strain collections, have facilitated a systems biology investigative strategy designed to enhance and accelerate our understanding of disease processes. Here, we provide examples of the progress made by exploiting an integrated genome-wide research platform to gain new insight into molecular pathogenesis. The studies have provided many new avenues for basic and translational research.
PMCID: PMC2735924  PMID: 19729843
16.  Molecular characterization of group A Streptococcus maltodextrin catabolism and its role in pharyngitis 
Molecular microbiology  2008;69(2):436-452.
We previously demonstrated that the cell-surface lipoprotein MalE contributes to GAS maltose/maltodextrin utilization, but MalE inactivation does not completely abrogate GAS catabolism of maltose or maltotriose. Using a genome-wide approach, we identified the GAS phosphotransferase system (PTS) responsible for non-MalE maltose/maltotriose transport. This PTS is encoded by an open reading frame (M5005_spy1692) previously annotated as ptsG based on homology with the glucose PTS in Bacillus subtilis. Genetic inactivation of M5005_spy1692 significantly reduced transport rates of radiolabeled maltose and maltotriose, but not glucose, leading us to propose its reannotation as malT for maltose transporter. The ΔmalT, ΔmalE, and ΔmalE:malT strains were significantly attenuated in their growth in human saliva and in their ability to catabolize α-glucans digested by purified human salivary α-amylase. Compared to wild-type, the three isogenic mutant strains were significantly impaired in their ability to colonize the mouse oropharynx. Finally, we discovered that the transcript levels of maltodextrin utilization genes are regulated by competitive binding of the maltose repressor MalR and catabolite control protein A. These data provide novel insights into regulation of the GAS maltodextrin genes and their role in GAS host-pathogen interaction, thereby increasing the understanding of links between nutrient acquisition and virulence in common human pathogens.
PMCID: PMC2761070  PMID: 18485073
Streptococcus; maltodextrin; transport; amylase; pharyngitis
17.  Sequential Outbreaks of Infections by Distinct Acinetobacter baumannii Strains in a Public Teaching Hospital in Houston, Texas▿  
Journal of Clinical Microbiology  2007;46(1):198-205.
Invasive disease due to Acinetobacter baumannii is an increasing problem in health care settings worldwide. Whether certain clones of A. baumannii are more likely to cause invasive disease in hospitalized patients is unknown. We studied all patients at a public teaching hospital in Houston, Texas, from whom the Acinetobacter calcoaceticus-Acinetobacter baumannii complex was isolated over a 14-month period in 2005 to 2006. One hundred seven unique patient isolates were identified, with 87 of the strains classified as being A. baumannii, the majority of which were multidrug resistant. The A. baumannii isolates were comprised of 18 unique pulsed-field types, with strains of clone A and clone B accounting for 66 of the 87 isolates. Epidemiologic analysis showed the predominance of the two A. baumannii clones at distinct time periods, with the remainder of the A. baumannii and non-A. baumannii strains being evenly distributed. Patients from whom clone A strains were isolated were more likely to be bacteremic than were patients with other A. baumannii isolates. Conversely, clone B strains were more likely to be isolated from patients with tertiary peritonitis. Patients from whom clone A was isolated had a significantly higher rate of mortality. Multilocus sequence typing demonstrated that clones A and B are related to each other and to A. baumannii strains previously isolated in Western Europe, sharing five of seven alleles. Taken together, we conclude that the outbreak of the A. calcoaceticus-A. baumannii complex in our institution was due to two distinct A. baumannii clones that were associated with significantly different patient outcomes.
PMCID: PMC2224270  PMID: 18003801
18.  Regulation of Polysaccharide Utilization Contributes to the Persistence of Group A Streptococcus in the Oropharynx▿  
Infection and Immunity  2007;75(6):2981-2990.
Group A Streptococcus (GAS) genes that encode proteins putatively involved in polysaccharide utilization show growth phase-dependent expression in human saliva. We sought to determine whether the putative polysaccharide transcriptional regulator MalR influences the expression of such genes and whether MalR helps GAS infect the oropharynx. Analysis of 32 strains of 17 distinct M protein serotypes revealed that MalR is highly conserved across GAS strains. malR transcripts were detectable in patients with GAS pharyngitis, and the levels increased significantly during growth in human saliva compared to the levels during growth in glucose-containing or nutrient-rich media. To determine if MalR influenced the expression of polysaccharide utilization genes, we compared the transcript levels of eight genes encoding putative polysaccharide utilization proteins in the parental serotype M1 strain MGAS5005 and its ΔmalR isogenic mutant derivative. The transcript levels of all eight genes were significantly increased in the ΔmalR strain compared to the parental strain, especially during growth in human saliva. Following experimental infection, the ΔmalR strain persistently colonized the oropharynx in significantly fewer mice than the parental strain colonized, and the numbers of ΔmalR strain CFU recovered were significantly lower than the numbers of the parental strain CFU recovered. These data led us to conclude that MalR influences the expression of genes putatively involved in polysaccharide utilization and that MalR contributes to the persistence of GAS in the oropharynx.
PMCID: PMC1932865  PMID: 17403878
19.  MalE of Group A Streptococcus Participates in the Rapid Transport of Maltotriose and Longer Maltodextrins▿ †  
Journal of Bacteriology  2007;189(7):2610-2617.
Study of the maltose/maltodextrin binding protein MalE in Escherichia coli has resulted in fundamental insights into the molecular mechanisms of microbial transport. Whether gram-positive bacteria employ a similar pathway for maltodextrin transport is unclear. The maltodextrin binding protein MalE has previously been shown to be key to the ability of group A Streptococcus (GAS) to colonize the oropharynx, the major site of GAS infection in humans. Here we used a multifaceted approach to elucidate the function and binding characteristics of GAS MalE. We found that GAS MalE is a central part of a highly efficient maltodextrin transport system capable of transporting linear maltodextrins that are up to at least seven glucose molecules long. Of the carbohydrates tested, GAS MalE had the highest affinity for maltotriose, a major breakdown product of starch in the human oropharynx. The thermodynamics and fluorescence changes induced by GAS MalE-maltodextrin binding were essentially opposite those reported for E. coli MalE. Moreover, unlike E. coli MalE, GAS MalE exhibited no specific binding of maltose or cyclic maltodextrins. Our data show that GAS developed a transport system optimized for linear maltodextrins longer than two glucose molecules that has several key differences from its well-studied E. coli counterpart.
PMCID: PMC1855798  PMID: 17259319
20.  Maltodextrin Utilization Plays a Key Role in the Ability of Group A Streptococcus To Colonize the Oropharynx  
Infection and Immunity  2006;74(8):4605-4614.
Analysis of multiple group A Streptococcus (GAS) genomes shows that genes encoding proteins involved in carbohydrate utilization comprise some 15% of the core GAS genome. Yet there is a limited understanding of how carbohydrate utilization contributes to GAS pathogenesis. Previous genome-wide GAS studies led us to a focused investigation of MalE, a putative maltodextrin-binding protein. Analysis of 28 strains of 22 distinct M protein serotypes showed that MalE is highly conserved among diverse GAS strains. malE transcript levels were significantly increased during growth in human saliva compared to growth in a chemically defined glucose-containing medium or a nutrient-rich medium. MalE was accessible to antibody binding, indicating that it is expressed on the GAS cell surface. Moreover, growth in human saliva appeared to increase MalE surface expression compared to growth in a nutrient-rich medium. Analysis of a ΔmalE isogenic mutant strain revealed decreased growth in human saliva compared to wild-type GAS. Radiolabeled carbohydrate binding assays showed that MalE was required for the binding of maltose but not glucose. The ΔmalE isogenic mutant strain colonized a lower percentage of GAS-challenged mice compared to wild-type and genetically complemented strains. Furthermore, decreased numbers of CFU were recovered from mice infected with the ΔmalE strain compared to those infected with wild-type GAS. These data demonstrate that maltodextrin acquisition is likely to be a key factor in the ability of GAS to successfully infect the oropharynx. Further investigation into carbohydrate transport and metabolism pathways may yield novel insights into GAS pathogenesis.
PMCID: PMC1539623  PMID: 16861648
21.  Growth Characteristics of and Virulence Factor Production by Group A Streptococcus during Cultivation in Human Saliva  
Infection and Immunity  2005;73(8):4723-4731.
Group A Streptococcus (GAS) commonly infects the human oropharynx, but the initial molecular events governing this process are poorly understood. Saliva is a major component of the innate and acquired immune defense in this anatomic site. Although landmark studies were done more than 60 years ago, investigation of GAS-saliva interaction has not been addressed extensively in recent years. Serotype M1 GAS strain MGAS5005 cultured in human saliva grew to ∼107 CFU/ml and, remarkably, maintained this density for up to 28 days. Strains of several other M-protein serotypes had similar initial growth patterns but did not maintain as high a CFU count during prolonged culture. As revealed by analysis of the growth of isogenic mutant strains, the ability of GAS to maintain high numbers of CFU/ml during the prolonged stationary phase in saliva was dependent on production of streptococcal inhibitor of complement (Sic) and streptococcal pyrogenic exotoxin B (SpeB). During cultivation in human saliva, GAS had growth-phase-dependent production of multiple proven and putative extracellular virulence factors, including Sic, SpeB, streptococcal pyrogenic exotoxin A, Mac protein, and streptococcal phospholipase A2. Our results clearly show that GAS responds in a complex fashion to growth in human saliva, suggesting that the molecular processes that enhance colonization and survival in the upper respiratory tract of humans are well under way before the organism reaches the epithelial cell surface.
PMCID: PMC1201272  PMID: 16040985
22.  In Vitro Killing of Community-Associated Methicillin-Resistant Staphylococcus aureus with Drug Combinations 
Antimicrobial Agents and Chemotherapy  2004;48(10):4016-4019.
This study employs time-kill techniques to examine the most common drug combinations used in the therapy of methicillin-resistant Staphylococcus aureus (MRSA) infections, vancomycin plus either gentamicin or rifampin. Community-associated MRSA were more likely to be synergistically inhibited by combinations of vancomycin and gentamicin versus vancomycin alone compared to inhibition associated with hospital-acquired strains.
PMCID: PMC521878  PMID: 15388469

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