Lateral gene transfer of streptococcal ICE element RD2 (region of difference 2) encoding secreted proteins

Background  

The genome of serotype M28 group A Streptococcus (GAS) strain MGAS6180 contains a novel genetic element named Region of Difference 2 (RD2) that encodes seven putative secreted extracellular proteins. RD2 is present in all serotype M28 strains and strains of several other GAS serotypes associated with female urogenital infections. We show here that the GAS RD2 element is present in strain MGAS6180 both as an integrative chromosomal form and a circular extrachromosomal element. RD2-like regions were identified in publicly available genome sequences of strains representing three of the five major group B streptococcal serotypes causing human disease. Ten RD2-encoded proteins have significant similarity to proteins involved in conjugative transfer of Streptococcus thermophilus integrative chromosomal elements (ICEs).     

Contribution of exogenous genetic elements to the group A Streptococcus metagenome

Variation in gene content among strains of a bacterial species contributes to biomedically relevant differences in phenotypes such as virulence and antimicrobial resistance. Group A Streptococcus (GAS) causes a diverse array of human infections and sequelae, and exhibits a complex pathogenic behavior. To enhance our understanding of genotype-phenotype relationships in this important pathogen, we determined the complete genome sequences of four GAS strains expressing M protein serotypes (M2, M4, and 2 M12) that commonly cause noninvasive and invasive infections. These sequences were compared with eight previously determined GAS genomes and regions of variably present gene content were assessed. Consistent with the previously determined genomes, each of the new genomes is approximately 1.9 Mb in size, with approximately 10% of the gene content of each encoded on variably present exogenous genetic elements. Like the other GAS genomes, these four genomes are polylysogenic and prophage encode the majority of the variably present gene content of each. In contrast to most of the previously determined genomes, multiple exogenous integrated conjugative elements (ICEs) with characteristics of conjugative transposons and plasmids are present in these new genomes. Cumulatively, 242 new GAS metagenome genes were identified that were not present in the previously sequenced genomes. Importantly, ICEs accounted for 41% of the new GAS metagenome gene content identified in these four genomes. Two large ICEs, designated 2096-RD.2 (63 kb) and 10750-RD.2 (49 kb), have multiple genes encoding resistance to antimicrobial agents, including tetracycline and erythromycin, respectively. Also resident on these ICEs are three genes encoding inferred extracellular proteins of unknown function, including a predicted cell surface protein that is only present in the genome of the serotype M12 strain cultured from a patient with acute poststreptococcal glomerulonephritis. The data provide new information about the GAS metagenome and will assist studies of pathogenesis, antimicrobial resistance, and population genomics.     

Secreted bacterial phospholipase A2 enzymes: better living through phospholipolysis

Phospholipases are ubiquitous and diverse enzymes that induce changes in membrane composition, activate the inflammatory cascade and alter cell signaling pathways. Recent evidence suggests that certain bacterial pathogens have acquired genes encoding secreted phospholipase A2 enzymes through lateral gene transfer events. The two best-studied members of this class of enzyme are ExoU and SlaA, which are produced by Pseudomonas aeruginosa and group A Streptococcus, respectively. These enzymes modulate the host inflammatory response, increase the severity of disease and otherwise alter host-pathogen interactions. We propose that a key function of ExoU and SlaA is to increase the fitness of the subclones expressing these enzymes, thereby increasing the population size of the PLA2-positive strains and enhancing the likelihood of encountering an at-risk host.     

The β-domain of cluster 2b streptokinase is a major determinant for the regulation of its plasminogen activation activity by cellular plasminogen receptors

Cluster 2b streptokinase (SK2b), secreted by invasive skin-trophic strains of Streptococcus pyogenes (GAS), is a human plasminogen (hPg) activator that optimally functions when human plasma hPg is bound, via its kringle-2 domain, to cognizant bacterial cells through the a1a2 domain of the major cellular hPg receptor, Plasminogen-binding group A streptococcal M-like protein (PAM). Another class of streptokinases (SK1), secreted primarily by GAS strains that possess affinity for pharyngeal infections, does not require PAM-bound hPg for optimal activity. We find herein that replacement of the central β-domain of SK2b with the same module from SK1 reduces the dependency of SK2b on PAM, and the converse is true when the β-domain of SK1 is replaced with this same region of SK2b. These data suggest that simple evolutionary shuttling of protein domains in GAS can be employed by GAS to rapidly generate strains that differ in tissue tropism and invasive capability and allow the bacteria to survive different challenges by the host.     

Expression of different group A streptococcal M proteins in an isogenic background demonstrates diversity in adherence to and invasion of eukaryotic cells

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M-like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein- and protein F1-deficient GAS strain, SAM2, which also lacks M-like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp-2 and HaCaT cells, a SAM2-derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp-2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.     

A novel,anchorless streptococcal surface protein that binds to human immunoglobulins

We have characterized a novel surface protein from urea extract of whole cells of group A Streptococcus pyogenes (GAS). A major protein band (35kD) was found to hybridize with human IgG by Western blotting. A search of the N-terminal amino acid sequence of this protein by using the GAS genome sequence database revealed an open reading frame that encoded a 38-kDa protein with a signal peptide sequence. We have named this protein streptococcal immunoglobulin-binding protein 35 (Sib35). It was found to be an anchorless protein with no LPXTG motif, distinct from the M protein superfamily exhibiting immunoglobulin-binding activity, and partially secreted in the culture supernatant. Recombinant Sib35 was also shown to bind human IgA and IgM. The sib35 gene was found in all GAS strains examined, but not in oral, group B, C, or G streptococcal strains. These results suggest that Sib35 is a unique immunoglobulin-binding protein in GAS.     

Regulatory rewiring confers serotype‐specific hyper‐virulence in the human pathogen group A Streptococcus

Phenotypic heterogeneity is commonly observed between isolates of a given pathogen. Epidemiological analyses have identified that some serotypes of the group A Streptococcus (GAS) are non‐randomly associated with particular disease manifestations. Here, we present evidence that a contributing factor to the association of serotype M3 GAS isolates with severe invasive infections is the presence of a null mutant allele for the orphan kinase RocA. Through use of RNAseq analysis, we identified that the natural rocA mutation present within M3 isolates leads to the enhanced expression of more than a dozen immunomodulatory virulence factors, enhancing phenotypes such as hemolysis and NAD⁺ hydrolysis. Consequently, an M3 GAS isolate survived human phagocytic killing at a level 13‐fold higher than a rocA complemented derivative, and was significantly more virulent in a murine bacteremia model of infection. Finally, we identified that RocA functions through the CovR/S two‐component system as levels of phosphorylated CovR increase in the presence of functional RocA, and RocA has no regulatory activity following covR or covS mutation. Our data are consistent with RocA interfacing with the CovR/S two‐component system, and that the absence of this activity in M3 GAS potentiates the severity of invasive infections caused by isolates of this serotype.     

Characterization of the immune response to collagen-like proteins Scl1 and Scl2 of serotype M1 and M28 group A Streptococcus

Group A Streptococcus (GAS) infections may trigger autoimmune sequelae thought to involve streptococcal antibodies cross-reactive to human antigens. Here, the antigenicity of the streptococcal collagen-like (CL) proteins, Scl1 and Scl2, that exhibit structural similarity to human collagen, was analyzed. Antibodies to Scl1.1 protein were detected in human sera from healthy individuals previously infected with M1 GAS and from patients with various GAS infections, as well as in sera from mice infected with M1 GAS. Linear B-cell epitope mapping identified immunoreactive peptides corresponding to the CL region of Scl1.1. Humoral responses to Scl1.28 and Scl2.28 were also detected in pediatric patients and mice infected with M28 GAS.     

Sortase A Induces Th17-Mediated and Antibody-Independent Immunity to Heterologous Serotypes of Group A Streptococci

Group A streptococci (GAS) are associated with a variety of mucosal and invasive human infections. Recurrent infections by highly heterologous serotypes indicate that cross-serotype immunity is critical for prevention of GAS infections; however, mechanisms underlying serotype-independent protection are poorly understood. Here we report that intranasal vaccination of mice with Sortase A (SrtA), a conserved cell wall bound protein, reduced colonization of nasal-associated lymphoid tissue (NALT) by heterologous serotypes of GAS. Vaccination significantly increased CD4⁺ IL-17A⁺ cells in NALT and depletion of IL-17A by neutralizing antibody prevented GAS clearance from NALT which was dependent on immunization with SrtA. Vaccination also induced high levels of SrtA-specific antibodies; however, immunized, B cell-deficient mice cleared streptococcal challenges as efficiently as wild type mice, indicating that the cross-serotype protection is Th17-biased and antibody-independent. Furthermore, efficient GAS clearance from NALT was associated with a rapid neutrophil influx into NALT of immunized mice. These results suggest that serotype independent immune protection against GAS mucosal infection can be achieved by intranasal vaccination with SrtA and enhanced neutrophil function is critical for anti-GAS defense and might be a target for prevention of GAS infections.     

Mutual Exclusivity of Hyaluronan and Hyaluronidase in Invasive Group A Streptococcus

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.     

A Bacterial Pathogen Co-opts Host Plasmin to Resist Killing by Cathelicidin Antimicrobial Peptides

The bacterial pathogen Group A Streptococcus (GAS) colonizes epithelial and mucosal surfaces and can cause a broad spectrum of human disease. Through the secreted plasminogen activator streptokinase (Ska), GAS activates human plasminogen into plasmin and binds it to the bacterial surface. The resulting surface plasmin protease activity has been proposed to play a role in disrupting tissue barriers, promoting invasive spread of the bacterium. We investigated whether this surface protease activity could aid the immune evasion role through degradation of the key innate antimicrobial peptide LL-37, the human cathelicidin. Cleavage products of plasmin-degraded LL-37 were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Ska-deficient GAS strains were generated by targeted allelic exchange mutagenesis and confirmed to lack surface plasmin activity after growth in human plasma or media supplemented with plasminogen and fibrinogen. Loss of surface plasmin activity left GAS unable to efficiently degrade LL-37 and increased bacterial susceptibility to killing by the antimicrobial peptide. When mice infected with GAS were simultaneously treated with the plasmin inhibitor aprotinin, a significant reduction in the size of necrotic skin lesions was observed. Together these data reveal a novel immune evasion strategy of the human pathogen: co-opting the activity of a host protease to evade peptide-based innate host defenses.     

The antibacterial properties of secreted phospholipases A2: a major physiological role for the group IIA enzyme that depends on the very high pI of the enzyme to allow penetration of the bacterial cell wall.

The antibacterial properties of human group IIA secreted phospholipase A(2) against Gram-positive bacteria as a result of membrane hydrolysis have been reported. Using Micrococcus luteus as a model system, we demonstrate the very high specificity of this human enzyme for such hydrolysis compared with the group IB, IIE, IIF, V, and X human secreted phospholipase A(2)s. A unique feature of the group IIA enzyme is its very high pI due to a large excess of cationic residues on the enzyme surface. The importance of this global positive charge in bacterial cell membrane hydrolysis and bacterial killing has been examined using charge reversal mutagenesis. The global positive charge on the enzyme surface allows penetration through the bacterial cell wall, thus allowing access of this enzyme to the cell membrane. Reduced bacterial killing was associated with the loss of positive charge and reduced cell membrane hydrolysis. All mutants were highly effective in hydrolyzing the bacterial membrane of cells in which the cell wall was permeabilized with lysozyme. These same overall characteristics were also seen with suspensions of Staphylococcus aureus and Listeria innocua, where cell membrane hydrolysis and antibacterial activity of human group IIA enzyme was also lost as a result of charge reversal mutagenesis.     

A Conserved UDP-Glucose Dehydrogenase Encoded outside the hasABC Operon Contributes to Capsule Biogenesis in Group A Streptococcus

Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major virulence factor, contributing to bloodstream survival through resistance to neutrophil and antimicrobial peptide killing and to in vivo pathogenicity. Capsule biosynthesis has been exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule abundance; however, a ΔhasB ΔhasB2 double mutant became completely acapsular. We conclude that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly identified HasB paralog, HasB2, which most likely resulted from gene duplication. The identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA capsule expression for M1T1 GAS pathogenicity and survival in the human host.     

Detection of 14-kDa group II phospholipase A2 in human seminal plasma

About 90% of phospholipase A2 activity detected in human seminal plasma reacted with monoclonal antibodies raised against human synovial fluid phospholipase A2. The crude seminal plasma yielded a pure immuno-cross-reactive phospholipase A2 preparation in a single purification step using immuno-affinity chromatography. The amino acid sequence of the N-terminal 20 residues of this seminal enzyme was determined and found to be identical with that of human synovial phospholipase A2. Thus, it is suggested that human seminal plasma contains phospholipase A2, belonging to the 14-kDa group II enzyme family, as the major isoenzyme.     

The fundamental contribution of phages to GAS evolution,genome diversification and strain emergence

The human bacterial pathogen group A Streptococcus (GAS) causes many different diseases including pharyngitis, tonsillitis, impetigo, scarlet fever, streptococcal toxic shock syndrome, necrotizing fasciitis and myositis, and the post-infection sequelae glomerulonephritis and rheumatic fever. The frequency and severity of GAS infections increased in the 1980s and 1990s, but the cause of this increase is unknown. Recently, genome sequencing of serotype M1, M3 and M18 strains revealed many new proven or putative virulence factors that are encoded by phages or phage-like elements. Importantly, these genetic elements account for an unexpectedly large proportion of the difference in gene content between the three strains. These new genome-sequencing studies have provided evidence that temporally and geographically distinct epidemics, and the complex array of GAS clinical presentations, might be related in part to the acquisition or evolution of phage-encoded virulence factors. We anticipate that new phage-encoded virulence factors will be identified by sequencing the genomes of additional GAS strains, including organisms non-randomly associated with particular clinical syndromes.     

A novel sortase, SrtC2, from Streptococcus pyogenes anchors a surface protein containing a QVPTGV motif to the cell wall

The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.     

In Vitro Activity of a New Antifungal Azolyl-substituted Indole Against Aspergillus fumigatus

A new 2- (α -azolylbenzyl)indole derivative exhibited high in vitro activity against 10 strains of Aspergillus fumigatus. This active compound, MT18n, had MIC of 2 μg/mL and is slightly less active than itraconazole and amphotericin B. The mechanism of action of this compound was evaluated through scanning electron microscopy, ergosterol biosynthesis inhibition and phospholipase A 2 -like activity inhibition studies. Scanning electron microscopy allowed observation of the membrane perturbations caused by MT18n and inference of a critical role of MT18n in membrane synthesis inhibition. Like other azole derivatives MT18n inhibits ergosterol biosynthesis, with a minimal inhibitory concentration of 6 μM. On the other hand, MT18n (10 μM) decreased the secreted phospholipase A 2 -like activity of Aspergillus fumigatus, an enzyme involved in the invasion process of the host. These results show the high in vitro activity of MT18n against Aspergillus fumigatus and suggest that this compound disturbs the membrane structure via ergosterol biosynthesis inhibition and exhibits phospholipase activity inhibition.