Magnus Rasmussen

SclA, a novel collagen-like surface protein of Streptococcus pyogenes

ABSTRACT Surface proteins of Streptococcus pyogenes are important virulence factors. Here we describe a novel collagen-like surface protein, designated SclA (streptococcal collagen-like surface protein). The sclA gene was identified in silico using the Streptococcal Genome Sequencing Project with the recently identified protein GRAB as the probe. SclA has a signal sequence and a cell wall attachment region containing the prototypic LPXTGX motif. The surface-exposed part of SclA contains a unique NH2-terminal domain of 73 amino acids, followed by a collagen-like region. ThesclA gene was found to be positively regulated by Mga, a transcriptional activator of several S. pyogenes virulence determinants. A mutant lacking cell wall-associated SclA was constructed and was found to be as effective as wild-type bacteria in platelet aggregation, survival in fresh human blood, and adherence to pharyngeal cells. The sclA gene was found in all 12S. pyogenes strains that were investigated using PCR. Sequence analysis revealed that the signal sequence and the cell wall attachment region are highly conserved. The collagen-like domain is variable in its NH2-terminal region and has conserved repeated domains in its COOH-terminal part. SclA proteins from most strains have additional proline-rich repeats spacing the collagen-like domain and the cell wall attachment sequence. The unique NH2-terminal region is hypervariable, but computer predictions indicate a common secondary structure, with two alpha helices connected by a loop region. Immune selection may explain the hypervariability in the NH2-terminal region, whereas the preserved secondary structure implies that this region has a common function. These features and the Mga regulation are shared with the M protein of S. pyogenes. Moreover, as with the gene encoding the M protein, phylogenetic analysis indicates that horizontal gene transfer has contributed to the evolution of sclA.

Biofilm formation by Propionibacterium acnes is a characteristic of invasive isolates

Propionibacterium acnes is a common and probably underestimated cause of delayed joint prosthesis infection. Bacterial biofilm formation is central in the pathogenesis of infections related to foreign material, and P. acnes has been shown to form biofilm both in vitro and in vivo. Here, biofilm formation by 93 P. acnes isolates, either from invasive infections (n = 45) or from the skin of healthy people (n = 48), was analysed. The majority of isolates from deep infections produced biofilm in a microtitre model of biofilm formation, whereas the skin isolates were poor biofilm producers (p <0.001 for a difference). This indicates a role for biofilm formation in P. acnes virulence. The type distribution, as determined by sequencing of recA, was similar among isolates isolated from skin and from deep infections, demonstrating that P. acnes isolates with different genetic backgrounds have pathogenic potential. The biofilm formed on plastic and on bone cement was analysed by scanning electron microscopy (EM) and by transmission EM. The biofilm was seen as a 10-mum-thick layer covering the bacteria and was composed of filamentous as well as more amorphous structures. Interestingly, the presence of human plasma in solution or at the plastic surface inhibits biofilm formation, which could explain why P. acnes primarily infect plasma-poor environments of, for example, joint prostheses and cerebrospinal shunts. This work underlines the importance of biofilm formation in P. acnes pathogenesis, and shows that biofilm formation should be considered in the diagnosis and treatment of invasive P. acnes infections.

Proteolysis and its regulation at the surface of Streptococcus pyogenes.

Pathogenic bacteria often produce proteinases that are believed to be involved in virulence. Moreover, several host defence systems depend on proteolysis, demonstrating that proteolysis and its regulation play an important role during bacterial infections. Here, we discuss how proteolytical events are regulated at the surface of Streptococcus pyogenes during infection with this important human pathogen.◊Streptococcus pyogenes produces proteinases, and host protein‐ases are produced and released as a result of the infection. Streptococcus pyogenes also recruits host proteinase inhibitors to its surface, suggesting that proteolysis is tightly regulated at the bacterial surface. We propose that the initial phase of a S. pyogenes infection is characterized by inhibition of proteolysis and complement activity at the bacterial surface. This is achieved mainly through binding of host proteinase inhibitors and complement regulatory proteins to bacterial surface proteins. In a later phase of the infection, massive proteolytic activity will release bacterial surface proteins and degrade human tissues, thus facilitating bacterial spread. These proteolytic events are regulated both temporally and spatially, and should influence virulence and the outcome of S. pyogenes infections.

Unique regulation of SclB – a novel collagen‐like surface protein of Streptococcus pyogenes

Slipped‐strand mispairing at sites containing so‐called coding repeats (CRs) can lead to phase variation of surface proteins in Gram‐negative bacteria. This mechanism, believed to contribute to virulence, has so far not been identified in a Gram‐positive bacterium. In the genome of the Gram‐positive human pathogen Streptococcus pyogenes, we identified pentanucleotide CRs within a putative signal sequence of an open reading frame (ORF) encoding a novel collagen‐like surface protein, denoted SclB. In 12 S. pyogenes strains, the number of CRs in the sclB gene varied from three to 19, rendering the start codon in frame with the downstream ORF in four strains and out of frame in eight strains. A protein reacting with anti‐SclB antibodies could only be solubilized from three strains, all containing an intact sclB gene. Variations in the number of CRs were observed within strains of the same M serotype and occurred during growth of S. pyogenes in fresh human blood, but not in medium. The SclB protein has a hypervariable N‐terminal part, a collagen‐like central part and a typical cell wall sorting sequence containing the LPXTGX motif. SclB is related to the collagen‐like SclA and is, like SclA, involved in the adhesion of S. pyogenes bacteria to human cells. However, the Mga protein, known to upregulate sclA and several additional genes encoding virulence factors of S. pyogenes, downregulates sclB transcription. This observation and the potential of SclB to phase vary by slipped‐strand mispairing emphasize the unique regulation of this novel S. pyogenes surface protein.

Large but not small copy-number alterations correlate to high-risk genomic aberrations and survival in chronic lymphocytic leukemia: a high-resolution genomic screening of newly diagnosed patients.

Large but not small copy-number alterations correlate to high-risk genomic aberrations and survival in chronic lymphocytic leukemia: a high-resolution genomic screening of newly diagnosed patients

α2-Macroglobulin-Proteinase Complexes Protect Streptococcus pyogenes from Killing by the Antimicrobial Peptide LL-37

The significant human bacterial pathogen Streptococcus pyogenes expresses GRAB, a surface protein that binds α2-macroglobulin (α2M), a major proteinase inhibitor of human plasma. α2M inhibits proteolysis by trapping the proteinase, which, however, still remains proteolytically active against smaller peptides that can penetrate the α2M-proteinase complex. Here we report that SpeB, a cysteine proteinase secreted by S. pyogenes, is trapped by α2M bound to protein GRAB. As a consequence, SpeB is retained at the bacterial surface and protects S. pyogenes against killing by the antibacterial peptide LL-37.

Improved Pattern for Genome-Based Screening Identifies Novel Cell Wall-Attached Proteins in Gram-Positive Bacteria

ABSTRACT With a large number of sequenced microbial genomes available, tools for identifying groups or classes of proteins have become increasingly important. Here we present an improved pattern for the identification of cell wall-attached proteins (CWPs), a group of proteins with diverse and important functions in gram-positive bacteria. This tripartite pattern is based on analysis of 65 previously described cell wall-attached proteins and takes into account the three principal requirements for cell wall sorting; a sortase target region (LPXTGX), a membrane-spanning region, and a charged stop-transfer tail. In five different genomes of gram-positive bacteria, the tripartite pattern identified a total of 35 putative CWPs, 19 of which were novel. The specificity and sensitivity of the tripartite pattern are higher than those of the classical pattern, which is based solely on the sortase target region. Several putative CWPs with atypical sortase target regions were identified. In the complete genome of the important human pathogen Streptococcus pyogenes, the tripartite pattern identified 14 putative CWPs. Seven of the putative S. pyogenes proteins were novel, and two of these were a 5′ nucleotidase and a pullulanase. This study represents the first whole-genome screening for CWPs, and we conclude that the tripartite pattern is highly suitable for this purpose. Identification of CWPs using this pattern offers important possibilities in the study of the pathogenesis and physiology of gram-positive bacteria.

Low Antibody Levels against Cell Wall-Attached Proteins of Streptococcus pyogenes Predispose for Severe Invasive Disease

Acute-phase serum samples from 70 patients with group A streptococcal (GAS) invasive disease were analyzed for IgG antibodies against 6 recently characterized GAS virulence factors (SclA, SclB, GRAB, MtsA, EndoS, and IdeS) and SpeB. Antibody levels against the cell wall-attached GAS antigens SclA, SclB, and GRAB were significantly lower in patients with severe invasive disease (streptococcal toxic shock syndrome [STSS] and/or necrotizing fasciitis [NF]; n=35), compared with levels in patients with nonsevere GAS bacteremia (n=35). Among patients with severe invasive disease, significantly lower antibody levels against GRAB were found in patients with STSS (n=10) than in patients with NF (n=17). Antibody levels against SpeB in patients with severe bacteremia were similar to those in patients with nonsevere bacteremia, and levels in patients with STSS were similar to those in patients with NF. The data indicate that immunity to cell wall-attached proteins may play a role in the protection against severe invasive disease and that antibodies against GRAB may be of importance in the pathogenesis of STSS.

Aerococci and aerococcal infections.

Aerococcus is a genus that comprises seven species, of which Aerococcus urinae, and Aerococcus sanguinicola are emerging human pathogens. Aerococci are gram positive cocci that are easily misidentified as streptococci or staphylococci, and thus the incidence of aerococcal infections has been underestimated. With the introduction of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) clinical microbiologists now have access to a rapid and accurate method to identify aerococci. A. urinae and A. sanguinicola are isolated in a small proportion of urinary specimens in many laboratories and many patients with bacteriuria with aerococci have symptoms of urinary tract infection (UTI). A. urinae, and also A. sanguinicola, cause invasive infections including infective endocarditis (IE) with many reported fatalities. Especially older men with urinary tract abnormalities are at risk for bacteraemia with A. urinae but the prognosis of bacteraemia without IE is favourable. Penicillin is appropriate for treatment of invasive infections and in IE, addition of an aminoglycoside should be considered. Treatment of UTI with aerococci is complicated by uncertainty about the effect of trimethoprim-sulphametoxazole and fluoroquinolones on aerococci. This review will discuss identification of Aerococcus spp., antibiotic resistance, the clinical presentation and management of aerococcal infections as well as the virulence mechanisms of these bacteria.

Clinical and microbiological features of bacteraemia with Aerococcus urinae.

Aerococcus urinae is a Gram-positive bacterium that can cause invasive infection, including infectious endocarditis (IE), mainly in older men. A. urinae is often misclassified in routine diagnostic laboratories. Through searches in the laboratory databases we identify 16 isolates of A. urinae causing bacteraemia during a 6-year period in southern Sweden, indicating that bacteraemia with A. urinae occurs in at least three cases per million inhabitants per year. The identity of isolates was confirmed by sequencing of the 16S rRNA genes and antibiotic susceptibility testing identified two ciprofloxacin-resistant isolates. A. urinae was the only significant pathogen isolated in all cases. Fifteen of the 16 patients were male, 15/16 were more than 70 years old, and 12/16 had underlying urological conditions. Though a urinary tract focus was suspected in the majority of cases, the bacterium was rarely found in urinary samples. Nine patients fulfilled the criteria for severe sepsis and an additional four fulfilled the criteria for sepsis. Only one fatality was recorded. Patients were treated mainly with beta-lactam antibiotics but fluoroquinolones and clindamycin were also used. Three cases of IE were diagnosed and these were complicated by spondylodiscitis in one case and by septic embolization to the brain in one case. An increased awareness of A. urinae is crucial to establishing its role as an important pathogen in older men with urinary tract disease.