Study On The Function Of The Membrane-bound Lytic Murein Transglycosylase A (MltA) In Edwardsiella Tarda

Edwardsiella tarda is a causative agent of edwardsiellosis with a wide host rangeincluding fish, amphibians, reptiles and mammalia, which has been the hazardousGram-negative bacteria in aquaculture industry and is responsible for major economiclosses worldwide. More importantly, Edw. tarda is a zoonotic infectious pathogenicbacteria which is the solely species of Edwardsiella genus that can cause infections inhuman, while relatively infrequent. The pathogenicity of E. tarda is polyfactorial andsome of the virulence properties have been implicated, including dermatotoxins, theheme-iron uptake system, haemolysins, phagocytic killing, a type III and a type VIsecretion system, the ability to produce biofilm, and invasion of epithelial cells.However, the major virulence factors of Edw. tarda are still poorly understood. Toprevent and treat edwardsiellosis, it is necessary to fully understand the pathogenicitymechanism of E. tarda.Lytic transglycosylases （LTs） are a class of autolysins, which are capable of cleavingthe β-1,4glycosidic bond between N-acetylmuramic acid and N-acetylglucosamineresidues of peptidoglycan, with concomitant formation of1,6-anhydromuramic acidresidues. Previous studies have arranged known and hypothetical LTs into fourfamilies based on amino acid sequence similarities and identified consensus motifs.However, the specific role of each family is largely unknown. By far, LTs have beensuggested to be involved in numerous cellular processes including cell growth andseparation, cell wall turnover, motility, drug resistance, protein secretion,differentiation and pathogenicity.The annotation of the virulent Edw. tarda EIB202genome indicates the presence of aputative membrane-bound lytic transglycosylase gene with the locus tag asETAE₀715. This gene is predicted to encode a383-amino-acid protein which has a3D domain at the C-terminal end with75%identity to the membrane-bound lytic murein transglycosylase A （MltA） in Esc. coli. Therefore, the gene was designated asmltA and the putative protein was predicted to function as lytic transglycosylase. Asan attempt to interpret the role of MltA in Edw. tarda, i) an mltA in-frame deletionmutant （ΔmltA） and an mltA overexpression strain （mltA⁺） of Edw. tarda EIB202were constructed; ii) compared ΔmltA and mltA⁺strains with the wild type strain interms of biological properties, antibiotic susceptibilities and virulence to fish.In this study, an mltA in-frame deletion mutant （ΔmltA） of E. tarda EIB202wasconstructed by overlap PCR and double crossover allelic exchange using suicidevector pRE118. Briefly, the upstream fragment and downstream part of mltA wereamplified by PCR with two primer pairs. The DNA fragment containing the ΔmltAwas amplified by overlap PCR. The fused segment （ΔmltA） was sequenced and thenligated into linear pRE118. The resulting plasmid was transformed into E. coli SY327and then introduced into E. coli SY17-1for mating into Edw. tarda EIB202byconjugation. After the plasmid pRE118-ΔmltA was integrated into the genomethrough a homologous recombination, the allelic exchange between the chromosomalgene and the in-frame deleted plasmidic copy was achieved in a second crossoverevent which was counter-selected on TSA containing10%sucrose to demonstrate theexcision of pRE118from the chromosome. In addition, an mltA overexpression Edw.tarda strain was constructed by electrotransforming a low copy plasmid carrying botha Kan resistance gene and an intact mltA gene harboring the putative promoter regioninto ΔmltA.The wild type strain of Edw. tarda displayed moderately increased amounts of MltAin the presence of ampicillin. However, mltA⁺exhibited sharply decreased amounts ofMltA when treated with ampicillin compared to that treated without ampicillin. Eitherinactivation or overexpression of MltA in Edw. tarda resulted in elevated sensitivityto β-lactam antibiotics and lower viability in oligotrophic or high osmoticenvironment than wild type strain. The wild type strain and ΔmltA represented similargrowth pattern, mltA⁺had a minor growth defect. Swimming motility assay showedthat mltA⁺exhibited an increased motility. By contrast, the ΔmltA mutant wasdefective for swimming motility. In vitro biofilm formation between the wild type strain and the mutants of Edw. tarda was significant difference. However, in vivobiofilm formation assay showed that the wild type strain and the mutants of Edw.tarda could not form observable biofilm on the surface of tissues in moribund fishinfected by Edw. tarda strains. Analysis of LPS showed that ΔmltA was defective inthe production of bands of low-molecular weight, while overexpression of mltA led toan increased generation of other two bands of low-molecular weight. The LPSinjection assay indicated that the increased generation of low-molecular weight LPSbands contributes to the pathogenicity of Edw. tarda. Moreover, the ΔmltA mutantexhibited a10-fold decrease （no significant difference） in virulence compared to thewild type strain, while the virulence of mltA⁺strain increased by～100-fold comparedto that of EIB202. The results indicated that MltA plays essential roles in β-lactamantibiotics and environmental stresses resistance, autolysis, LPS biosynthesis, andpathogenicity of E. tarda. This is the first report that MltA has a virulence-relatedfunction in E. tarda. This study provided useful information for further studies onpathogenesis of E. tarda.