Sialic Acid-Mediated Gene Expression in Streptococcus pneumoniae and Role of NanR as a Transcriptional Activator of the nan Gene Cluster

Abstract

In this study, we investigated the transcriptomic response of Streptococcus pneumoniae D39 to sialic acid (N-acetylneuraminic acid [Neu5Ac]). Transcriptome comparison of wild-type D39 grown in M17 medium with and without sialic acid revealed the elevated expression of various genes and operons, including the nan gene cluster (nan operon I and nanA gene). Our microarray analysis and promoter-lacZ fusion studies showed that the transcriptional regulator NanR acts as a transcriptional activator of nan operon I and the nanA gene in the presence of sialic acid. The putative regulatory site of NanR in the promoter region of nan operon I is predicted and confirmed by promoter truncation experiments. Furthermore, the role of CcpA in the regulation of the nan gene cluster is demonstrated through microarray analysis and promoter-lacZ fusion studies, suggesting that in the presence of sialic acid and glucose, CcpA represses the expression of nan operon I while the expression of the nanA gene is CcpA independent.T he low-GC Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen and the causal agent of many diseases, including pneumonia, sepsis, meningitis, otitis media, and conjunctivitis, which result in over a million deaths each year worldwide (1, 2). S. pneumoniae colonizes the human nasopharynx during the first few months of life (3). For survival in the host, bacteria rely not only on the different virulence factors they possess but also on the appropriate use of nutrients available in their habitat (4, 5). The ability of S. pneumoniae to utilize a variety of carbohydrate sources is one of the crucial factors in successful colonization and in causing pneumococcal infections (6). Metabolic selection enables a bacterium to choose a preferred source of carbon over a nonpreferred one through a mechanism called carbon catabolite repression (CCR) (5). CcpA (carbon catabolite protein A) is a transcription factor that mediates CCR in the presence of a preferred sugar source, e.g., glucose, by binding to catabolite repression elements (cre boxes) found in the promoter regions of CcpA-targeted genes (5, 7-9). Despite the importance of carbohydrates in the pathogenesis of S. pneumoniae, research concerning metabolic pathways of S. pneumoniae still demands more attention.Sialic acid is one of the most important carbohydrates for S. pneumoniae, since it plays a vital role as a carbon/energy source, a receptor for adhesion and invasion, and a molecular signal for the promotion of biofilm formation, nasopharyngeal carriage, and invasion of the lungs (10). It has been shown that S. pneumoniae can utilize sialic acid as a carbon source, which results in improved pneumococcal biofilm formation in vitro, at concentrations comparable to those of free sialic acid in human saliva (11,12). Of the 43 known naturally occurring derivatives of the nine-carbon sugar neuraminic acid, N-acetylneuraminic acid (Neu5Ac) is the only one found in humans (13). Another form of sialic acid is N-glycolylneuraminic acid (Neu5Gc), which i...

Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization

“…For this reason, a major part of the pneumococcus genome is dedicated toward the uptake and processing of carbohydrates, including sialic acid, galactose, and glucose (23,52). Since the initial discovery that the pneumococcus produces neuraminidase, much effort has gone toward elucidating its role in the disease process (30,34,35,53,54), determining the impact of free sialic acid on the bacterium's metabolism (31,33,55,56), and testing whether inhibition of neuraminidase activity is protective (57). Along such lines, Marion et al determined that S. pneumoniae was capable of growth with sialic acid as the sole carbon source (33).…”

Streptococcus pneumoniae Senses a Human-like Sialic Acid Profile via the Response Regulator CiaR

“…The nanAB locus contains genes encoding sialidases, such as NanA, and the main Sia transporter SatABC (King et al, 2004; Marion et al, 2011). Furthermore, the locus contains catabolite repression elements (cre), leading to transcriptional inhibition of genes within the locus in the presence of glucose (Afzal et al, 2015; Gualdi et al, 2012). NanA scavenges Sias from host glycoconjugates (King et al, 2006; Tong et al, 2002), and is a known pneumococcal virulence factor promoting colonization of the nasopharynx and lungs (Orihuela et al, 2004), invasion of the brain endothelium (Uchiyama et al, 2009), cytokine release in human monocytes, and NET formation of human neutrophils (Chang et al, 2012).…”

Transcription of Sialic Acid Catabolism Genes in Corynebacterium glutamicum Is Subject to Catabolite Repression and Control by the Transcriptional Repressor NanR

“…Besides serving as attachment and recognition point for various pathogens, Neu5Ac is also an important source of carbon and energy available in various host niches such as the oral cavity and the respiratory, intestinal, and urogenital tracts (29)(30)(31)(32). Therefore, Neu5Ac metabolism and its control have been studied in detail for various, often pathogenic, bacteria such as Escherichia coli, Vibrio vulnificus, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Clostridium perfringens, and the probiotic Bifidobacterium breve (33)(34)(35)(36)(37)(38). In H. influenzae, transcription of the nan and the siaPT operons for Neu5Ac utilization is repressed by SiaR and is activated in the presence of glucosamine 6-phosphate (see Table S1 in the supplemental material), which is an intermediate of Neu5Ac catabolism and acts as a coactivator of the RpiR-type regulator SiaR (37,(39)(40)(41).…”

“…Binding of the Neu5Ac catabolism intermediate N-acetylmannosamine-6-phosphate (ManNAc-6P) to V. vulnificus NanR mediates relocation of residues in the ligand binding site, thus alleviating the interaction between the NanR dimer and DNA and subsequently relieving the repression by NanR and inducing transcription of the nan operons (42) (see Table S1). Different from the RpiR-type regulators of Neu5Ac metabolism of H. influenzae and V. vulnificus, the RpiR-type regulator NanR of S. pneumoniae acts as a transcriptional activator of Neu5Ac catabolism genes (34). In E. coli and B. breve, transcriptional control of genes for Neu5Ac utilization is brought about by GntR-type transcriptional repressors each also named NanR, which both depend on Neu5Ac as the sole inducer (44-46) (see Table S1).…”