Lysozyme Resistance in Streptococcus suis Is Highly Variable and Multifactorial

Background: Streptococcus suis is an important infectious agent for pigs and occasionally for humans. The host innate immune system plays a key role in preventing and eliminating S. suis infections. One important constituent of the innate immune system is the protein lysozyme, which is present in a variety of body fluids and immune cells. Lysozyme acts as a peptidoglycan degrading enzyme causing bacterial lysis. Several pathogens have developed mechanisms to evade lysozyme-mediated killing. In the present study we compared the lysozyme sensitivity of various S. suis isolates and investigated the molecular basis of lysozyme resistance for this pathogen. Results: The lysozyme minimal inhibitory concentrations of a wide panel of S. suis isolates varied between 0.3 to 10 mg/ml. By inactivating the oatA gene in a serotype 2 and a serotype 9 strain, we showed that OatA-mediated peptidoglycan modification partly contributes to lysozyme resistance. Furthermore, inactivation of the murMN operon provided evidence that additional peptidoglycan crosslinking is not involved in lysozyme resistance in S. suis. Besides a targeted approach, we also used an unbiased approach for identifying factors involved in lysozyme resistance. Based on whole genome comparisons of a lysozyme sensitive strain and selected lysozyme resistant derivatives, we detected several single nucleotide polymorphisms (SNPs) that were correlated with the lysozyme resistance trait. Two SNPs caused defects in protein expression of an autolysin and a capsule sugar transferase. Analysis of specific isogenic mutants, confirmed th

Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureus

The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling

Initial Characterization of the FlgE Hook High Molecular Weight Complex of

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility

Antimicrobial resistance data on 16,756 Streptococcus pneumoniae isolates in 1999: A pan-regional multicenter surveillance study in France

International audienc

Bacillus subtilis σV Confers Lysozyme Resistance by Activation of Two Cell Wall Modification Pathways, Peptidoglycan O-Acetylation and d-Alanylation of Teichoic Acids ▿ †

The seven extracytoplasmic function (ECF) sigma (σ) factors of Bacillus subtilis are broadly implicated in resistance to antibiotics and other cell envelope stressors mediated, in part, by regulation of cell envelope synthesis and modification enzymes. We here define the regulon of σV as including at least 20 operons, many of which are also regulated by σM, σX, or σW. The σV regulon is strongly and specifically induced by lysozyme, and this induction is key to the intrinsic resistance of B. subtilis to lysozyme. Strains with null mutations in either sigV or all seven ECF σ factor genes (Δ7ECF) have essentially equal increases in sensitivity to lysozyme. Induction of σV in the Δ7ECF background restores lysozyme resistance, whereas induction of σM, σX, or σW does not. Lysozyme resistance results from the ability of σV to activate the transcription of two operons: the autoregulated sigV-rsiV-oatA-yrhK operon and dltABCDE. Genetic analyses reveal that oatA and dlt are largely redundant with respect to lysozyme sensitivity: single mutants are not affected in lysozyme sensitivity, whereas an oatA dltA double mutant is as sensitive as a sigV null strain. Moreover, the sigV oatA dltA triple mutant is no more sensitive than the oatA dltA double mutant, indicating that there are no other σV-dependent genes necessary for lysozyme resistance. Thus, we suggest that σV confers lysozyme resistance by the activation of two cell wall modification pathways: O-acetylation of peptidoglycan catalyzed by OatA and d-alanylation of teichoic acids by DltABCDE

Bacterial pathogenomics.

Genomes from all of the crucial bacterial pathogens of humans, plants and animals have now been sequenced, as have genomes from many of the important commensal, symbiotic and environmental microorganisms. Analysis of these sequences has revealed the forces that shape pathogen evolution and has brought to light unexpected aspects of pathogen biology. The finding that horizontal gene transfer and genome decay have key roles in the evolution of bacterial pathogens was particularly surprising. It has also become evident that even the definitions for 'pathogen' and 'virulence factor' need to be re-evaluated