Deacetylation of sialic acid by esterases potentiates pneumococcal neuraminidase activity for mucin utilization, colonization and virulence

Abstract

Pneumococcal neuraminidase is a key enzyme for sequential deglycosylation of host glycans, and plays an important role in host survival, colonization, and pathogenesis of infections caused by Streptococcus pneumoniae. One of the factors that can affect the activity of neuraminidase is the amount and position of acetylation present in its substrate sialic acid. We hypothesised that pneumococcal esterases potentiate neuraminidase activity by removing acetylation from sialic acid, and that will have a major effect on pneumococcal survival on mucin, colonization, and virulence. These hypotheses were tested using isogenic mutants and recombinant esterases in microbiological, biochemical and in vivo assays. We found that pneumococcal esterase activity is encoded by at least four genes, SPD_0534 (EstA) was found to be responsible for the main esterase activity, and the pneumococcal esterases are specific for short acyl chains. Assay of esterase activity by using natural substrates showed that both the Axe and EstA esterases could use acetylated xylan and Bovine Sub-maxillary Mucin (BSM), a highly acetylated substrate, but only EstA was active against tributyrin (triglyceride). Incubation of BSM with either Axe or EstA led to the acetate release in a time and concentration dependent manner, and pre-treatment of BSM with either enzyme increased sialic acid release on subsequent exposure to neuraminidase A. qRT-PCR results showed that the expression level of estA and axe increased when exposed to BSM and in respiratory tissues. Mutation of estA alone or in combination with nanA (codes for neuraminidase A), or the replacement of its putative serine active site to alanine, reduced the pneumococcal ability to utilise BSM as a sole carbon source, sialic acid release, colonization, and virulence in a mouse model of pneumococcal pneumonia.

Glycan‐metabolizing enzymes in microbe–host interactions: the Streptococcus pneumoniae paradigm

“…The crystal structure of EstA reveals a dimeric organization that is stabilized by the presence of a calcium ion. Three other esterases have been identified in S. pneumoniae, but only EstA was found to contribute to the growth of the bacterium on BSM [89]. Three other esterases have been identified in S. pneumoniae, but only EstA was found to contribute to the growth of the bacterium on BSM [89].…”

“…However, there is also a wealth of evidence to show that the role of Nglycan degradation in the host-pathogen interaction extends beyond nutrition. NanA, as the initiator of complex N-glycan degradation, has been identified as a key virulence factor in S. pneumoniae that contributes to adherence [69,78,[80][81][82], biofilm formation [81][82][83][84], tissue invasion [69,85], colonization [82,[86][87][88][89], persistence [86,87], immune Box 2. NanA, as the initiator of complex N-glycan degradation, has been identified as a key virulence factor in S. pneumoniae that contributes to adherence [69,78,[80][81][82], biofilm formation [81][82][83][84], tissue invasion [69,85], colonization [82,[86][87][88][89], persistence [86,87], immune Box 2.…”

“…Adherence is considered an important contributor to the ability of S. pneumoniae to colonize the human airway [92]. Therefore, given the evidence to suggest that NanA contributes to pneumococcal adherence, it is not surprising that the majority of animal model studies performed with nanA mutants have reported reduced colonization rates [86][87][88][89]. Two of these studies also observed an effect of NanA on pneumococcal persistence [86,87], which may relate to its ability to contribute to opsonophagocytic killing resistance [90].…”

Assessing the function of pneumococcal neuraminidases NanA, NanB and NanC inin vitroandin vivolung infection models using monoclonal antibodies

“…Previous studies reported a substantially reduced bacterial burden in the lungs of mice infected intranasally with nan A and/or nan B gene deletion mutant strains and improved host survival [39,40,68]. NanA was shown to contribute to nasopharyngeal colonisation [41,43,68–70].…”

Identification and Crystallographic Analysis of a New Carbohydrate Acetylesterase (SmAcE1) from Sinorhizobium meliloti

“…Bacterial CEs have important physiological functions, which were proven by that the CE mutant shows growth retardation [19,20]. However, only few bacterial CEs have been identified and investigated in detail.…”