Point mutations in wchA are responsible for the non-typability of two invasive Streptococcus pneumoniae isolates

Abstract

Non-typable Streptococcus pneumoniae (NTPn) strains are typically isolated from nasopharyngeal carriage or from conjunctivitis. Since the isolation of NTPn from invasive disease is rare, we characterized the genetic basis of the non-typability of two isolates obtained in Italy from two cases of bacteraemic pneumonia. MLST revealed that both NTPn belonged to ST191, which, according to the MLST database, is associated with serotype 7F. Sequencing of the capsular locus (cps) confirmed the presence of a 7F cps in both strains and revealed the existence of distinct single point mutations in the wchA gene (a glycosyltransferase), both leading to the translation of proteins truncated at the C terminus. To verify that these mutations were responsible for the non-typability of the isolates, a functional 7F WchA was overexpressed in both NTPn. The two NTPn along with their WchA-overexpressing derivatives were analysed by transmission electron microscopy and by high-resolution magic angle spinning NMR spectroscopy. Both NTPn were devoid of a polysaccharide capsule, and WchA overexpression was sufficient to restore the assembly of a serotype 7F capsule on the surface of the two NTPn. In conclusion, we identified two new naturally occurring point mutations that lead to non-typability in the pneumococcus, and demonstrated that WchA is essential for the biosynthesis of the serotype 7F capsule. INTRODUCTIONThe Gram-positive pathogen Streptococcus pneumoniae is a major cause of community-acquired pneumonia, as well as upper respiratory tract infections such as acute otitis media and sinusitis, and invasive diseases such as meningitis, bacteraemia and endocarditis. However, S. pneumoniae is also a commensal of the upper respiratory tract, especially of young children, who represent the reservoir for pneumococcal transmission within the community.The ability of the pneumococcus to invade sterile sites is related to the expression of a polysaccharide capsule, which provides a barrier against host cell-mediated phagocytosis and allows bacterial persistence in the blood (Brown et al., 1983). In addition, capsular polysaccharides (CPSs) are immunogenic and antibodies against CPSs provide protection against pneumococcal disease; therefore, all of the current pneumococcal vaccines are based on a combination of different CPSs, either unconjugated or conjugated to a carrier protein (Gladstone et al., 2011).To date, 93 capsular types (serotypes) have been described on the basis of their different genetic, biochemical and antigenic properties (Bentley et al., 2006;Calix & Nahm, 2010;Park et al., 2007). Each pneumococcal serotype corresponds to a distinct CPS, synthesized on the bacterial surface by enzymes which are encoded within a single capsule locus (cps), with a few notable exceptions (Llull et al., 1999). The sequencing of different cps loci has revealed the presence of a core set of genes common to all capsular types, namely wzg, wzh, wzd and wze (also known as cpsA, B, C and D), and of additional genes that are CPS-specific (Bentley...

Streptococcus pseudopneumoniae as an emerging pathogen from patients with respiratory diseases

“…There were three isolates of the examined S. pneumoniae found to be positive for aliB-like ORF2 gene; our results are in agreement with previous findings that have recorded the same observation (Simo˜es et al, 2011) which could be explained by the lack of ability of those isolates to produce the capsule in vitro (Scott et al, 2012) as a result of capsular genes mutation (Melchiorre et al, 2012).…”

Pneumococcal Capsules and Their Types: Past, Present, and Future

“…These proteins can be used to classify group II NTs into three clades called null capsule clades (NCCs) (111). In contrast, group I NT isolates almost always have cps locus defects, found mostly in wchA/cpsE (112,114), and for unknown reasons, serotype 8 is the most prone to becoming NT (112). In two recent studies, no NT strains with an intact cps locus that could potentially synthesize a wholly novel capsular PS were identified (100, 112), although one study found a serologically ambiguous strain possibly representing a novel member of serogroup 33 (100) exhibiting 99% sequence homology to partial sequences from a "serotype 33E" cps locus reported in GenBank (accession no.…”

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

“…When expressed in E. coli, this gene is sufficient to synthesize a glucose-P-lipid intermediate (70). Deletion of this gene in S. pneumoniae strains resulted in failure to elaborate a capsule (71). S. aureus capM encodes a protein with homology to WchA, and may serve as the membrane-bound sugar transferase for the synthesis of the UDP-FucNAc linkage (Fig.…”