Effects of inactivation of d,d -transpeptidases of Acinetobacter baumannii on bacterial growth and susceptibility to β-lactam antibiotics

Resistance to β-lactams, the most used antibiotics worldwide, constitutes the major problem for treatment of bacterial infections. In the nosocomial pathogen Acinetobacter baumannii , β-lactamase-mediated resistance to the family of β-lactam antibiotics, carbapenems, has resulted in the selection and dissemination of multidrug-resistant isolates, which often cause infections characterized by high mortality rates. There is thus an urgent demand for new β-lactamase-resistant antibiotics that also inhibit their targets, penicillin-binding proteins (PBPs). As some PBPs are indispensable for biosynthesis of the bacterial cell wall and survival, we evaluated their importance for growth of A. baumannii by performing gene inactivation studies of d,d- transpeptidase domains of high-molecular mass (HMM) PBPs individually and in combination with one another. We showed that PBP3 is essential for A. baumannii survival, as deletion mutants of this d,d- transpeptidase were not viable. Inactivation of PBP1a resulted in partial cell lysis and retardation of bacterial growth, and these effects were further enhanced by additional inactivation of PBP2 but not PBP1b. Susceptibility to β-lactam antibiotics increased 4-8-fold for the A. baumannii PBP1a/PBP1b/PBP2 triple mutant and 2-4-fold for all remaining mutants. Analysis of peptidoglycan structure revealed a significant change in the muropeptide composition of the triple mutant and demonstrated that lack of d,d- transpeptidase activity of PBP1a, PBP1b, and PBP2 is compensated by an increase in l,d- transpeptidase-mediated crosslinking activity of LdtJ. Overall, our data showed that in addition to essential PBP3, simultaneous inhibition of PBP1a and PBP2 or PBPs in combination with LdtJ could represent potential strategies for design of novel drugs against A. baumannii .

PGfinder, a novel analysis pipeline for the consistent, reproducible and high- resolution structural analysis of bacterial peptidoglycans

Many software solutions are available for proteomics and glycomics studies, but none are ideal for the structural analysis of peptidoglycan, the essential and major component of bacterial cell envelopes. It is comprised of glycan chains and peptide stems, both containing unusual amino acids and sugars. This has forced the field to rely on manual analysis approaches, which are time-consuming, labour-intensive, and prone to error. The lack of automated tools has hampered the ability to perform high-throughput analyses and prevented the adoption of a standard methodology. Here, we describe a novel tool called PGfinder for the analysis of peptidoglycan structure and demonstrate that it represents a powerful tool to quantify PG fragments and discover novel structural features. Our analysis workflow, which relies on open-access tools, is a breakthrough towards a consistent and reproducible analysis of bacterial peptidoglycans. It represents a significant advance towards peptidoglycomics as a full-fledged discipline.

AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin

Clostridium difficile 630 possesses a cryptic but functional gene cluster vanG Cd homologous to the vanG operon of Enterococcus faecalis . Expression of vanG Cd in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the meso-DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named asnB and asnB2 in the C. difficile genome whose products were potentially involved in this peptidoglycan structure modification. We found that asnB expression was only induced when C. difficile was grown in the presence of vancomycin, yet independently from the vanG Cd resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when asnB was inactivated. No change in vancomycin MIC was observed in the asnB mutant strain. In contrast, overexpression of asnB resulted in the amidation of most of the C. difficile peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in E. coli . In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of C. difficile in the presence of vancomycin.

Xylanimonas allomyrinae sp. nov. isolated from the gut of larva of Allomyrina dichotoma, reclassification of Xylanibacterium ulmi as Xylanimonas ulmi comb. nov. and Xylanimicrobium pachnodae as Xylanimonas pachnodae comb. nov., and emendation of the genus Xylanimonas

A bacterium that was Gram-staining-positive, facultatively anaerobic, non-motile, rod- or filamentous-shaped, designated as strain 2JSPR-7T, was isolated from a gut of larvae of Allomyrina dichotoma which were raised at the National Institute of Agricultural Sciences, Wanju-gun, Republic of Korea. 2JSPR-7T had the highest 16S rRNA gene sequence similarity to Xylanibacterium ulmi XIL08T (98.1 %), Xylanimicrobium pachnodae NBRC 107786T (97.8 %) and Xylanimonas cellulosilytica DSM 15894T (97.5 %). Optimum growth conditions were at 28–30 °C, pH 7–8 and 0 % salt concentration. The cellular fatty acids mainly consisted of anteiso-C15 : 0, C14 : 0 and C16 : 0. The polar lipids were diphosphatidylglycerol, four unidentified phospholipids and two unidentified glycophospholipids. The major menaquinones were MK-8(H4) and MK-9(H4). The peptidoglycan structure was suggested to be the type A3α (A11.14) l-Lys–l-Ser with the presence of d-Ala, l-Ala, d-Glu, l-Ser and l-Lys. Whole cell sugars were rhamnose, ribose and glucose. The DNA G+C content was 72.7 mol%. We encountered difficulty in selecting a suitable genus to accommodate strain 2JSPR-7T from any of the genera Xylanimonas , Xylanimicrobium and Xylanibacterium based on the polyphasic approach including phylogenetic and phenotypic characterization. Therefore, it is proposed to combine the genera Xylanimicrobium and Xylanibacterium with the genus Xylanimonas considering the priority of publication and to classify strain 2JSPR-7T in the genus as Xylanimonas allomyrinae sp. nov. The type strain of the novel species is 2JSPR-7T (=KACC 19330T=NBRC 113052T). In addition, the description of the genus Xylanimonas is emended, and Xylanibacterium ulmi and Xylanimicrobium pachnodae are reclassified as Xylanimonas ulmi comb. nov. and Xylanimonas pachnodae comb. nov., respectively.