Abstract
Mucoviscosity-associated gene A (magA) of Klebsiella pneumoniae contributes to K1 capsular polysaccharide (CPS) biosynthesis. Based on sequence homology and gene alignment, the magA gene has been predicted to encode a Wzy-type CPS polymerase. Sequence alignment with the Wzy_C and RfaL protein families (which catalyze CPS or lipopolysaccharide (LPS) biosynthesis) and topological analysis has suggested that eight highly conserved residues, including G308, G310, G334, G337, R290, P305, H323, and N324, were located in a hypothetical loop region. Therefore, we used site-directed mutagenesis to study the role of these residues in CPS production, and to observe the consequent phenotypes such as mucoviscosity, serum and phagocytosis resistance, and virulence (as assessed in mice) in pyogenic liver abscess strain NTUH-K2044. Alanine substitutions at R290 or H323 abolished all of these properties. The G308A mutant was severely impaired for these functions. The G334A mutant remained mucoid with decreased CPS production, but its virulence was significantly reduced in vivo. No phenotypic change was observed for strains harboring magA G310A, G337A, P305A, or N324A mutations. Therefore, R290, G308, H323, and G334 are functionally important residues of the MagA (Wzy) protein of K. pneumoniae NTUH-K2044, capsular type K1. These amino acids are also likely to be important for the function of Wzy in other capsular types in K. pneumoniae and other species bearing Wzy_C family proteins.
Highlights
Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that causes urinary tract infections, nosocomial pneumonia, and intra-abdominal infections [1]
We found that Mucoviscosity-associated gene A (magA)-adjacent sequences encoded proteins homologous to capsular polysaccharide (CPS) biosynthetic machinery; deletion/complementation experiments proved that magA was an essential gene for K1 CPS biosynthesis [7]
We identified the conserved amino acids of MagA and used Ala substitutions to analyze the role of these residues in CPS biosynthesis in the pyogenic liver abscess (PLA) strain NTUH-K2044
Summary
Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that causes urinary tract infections, nosocomial pneumonia, and intra-abdominal infections [1]. A new type of invasive K. pneumoniae disease has emerged worldwide as the source of community-acquired pyogenic liver abscess (PLA), especially in Asia [2,3,4,5]. This disease often is complicated by metastatic infections such as meningitis and endophthalmitis. The magA (mucoviscosityassociated gene A) was identified based on its role in mucoviscosity, resistance to serum killing and phagocytosis, and virulence in mice [6]. Based on limited sequences similarities, MagA was proposed to be an O-antigen ligase [6]. The mucoviscosity is indirectly related to magA because of its essential role in capsule production, whereas the mucoviscosity might be mediated by capsule expression promoting regulators such as rmpA [8,9]
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