1887

Abstract

Bacillus anthracis, an aetiologic agent of the zoonotic disease anthrax, encodes a putative NlpC/P60 endopeptidase BAS1812. It harbours a signal peptide, three bacterial SH3 domains and an NlpC/P60 family domain. Previous studies showed that BAS1812 is immunogenic in infected hosts and is a potential biomarker for anthrax treatment. To date, however, little information is known about its function and involvement in anthrax pathogenesis. Here we describe the phenotypic effect of BAS1812 deletion in B. anthracis Sterne strain. Transcriptional analysis showed that BAS1812 expression in a host-like environment was enhanced at the end of log phase, started to diminish after entry to stationary phase and increased again late in stationary phase. The constructed BAS1812 mutant showed impaired long-term survival in the stationary growth phase, less resilience to detergent, lesser endospore formation and delayed germination. The mutant also showed diminished ability to degrade peptidoglycan, but its ability to produce anthrax exotoxins was not affected. We hypothesize that BAS1812 is a cell wall hydrolase involved in biological activities related to maintaining cell wall integrity, sporulation and spore germination.

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2017-02-21
2019-10-20
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References

  1. Moat AG, Foster JW, Spector MP. Cell structure and function. In: Microbial Physiology John Wiley & Sons, Inc; 2002; pp.277–349[CrossRef]
    [Google Scholar]
  2. Born TL, Blanchard JS. Structure/function studies on enzymes in the diaminopimelate pathway of bacterial cell wall biosynthesis. Curr Opin Chem Biol 1999;3:607–613[PubMed][CrossRef]
    [Google Scholar]
  3. Heijenoort JV. Formation of the glycan chains in the synthesis of bacterial peptidoglycan. Glycobiology 2001;11:25R–36[PubMed][CrossRef]
    [Google Scholar]
  4. Kuhn M, Goebel W. Identification of an extracellular protein of Listeria monocytogenes possibly involved in intracellular uptake by mammalian cells. Infect Immun 1989;57:55–61[PubMed]
    [Google Scholar]
  5. Smith TJ, Blackman SA, Foster SJ. Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. Microbiology 2000;146:249–262 [CrossRef][PubMed]
    [Google Scholar]
  6. Anantharaman V, Aravind L. Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes. Genome Biol 2003;4:R11[PubMed][CrossRef]
    [Google Scholar]
  7. Haiser HJ, Yousef MR, Elliot MA. Cell wall hydrolases affect germination, vegetative growth, and sporulation in Streptomyces coelicolor. J Bacteriol 2009;191:6501–6512 [CrossRef][PubMed]
    [Google Scholar]
  8. Parthasarathy G, Lun S, Guo H, Ammerman NC, Geiman DE et al. Rv2190c, an NlpC/P60 family protein, is required for full virulence of Mycobacterium tuberculosis. PLoS One 2012;7:e43429 [CrossRef][PubMed]
    [Google Scholar]
  9. Mock M, Fouet A. Anthrax. Annu Rev Microbiol 2001;55:647–671 [CrossRef][PubMed]
    [Google Scholar]
  10. Tournier JN, Ulrich RG, Quesnel-Hellmann A, Mohamadzadeh M, Stiles BG. Anthrax, toxins and vaccines: a 125-year journey targeting Bacillus anthracis. Expert Rev Anti Infect Ther 2009;7:219–236 [CrossRef][PubMed]
    [Google Scholar]
  11. Jang J, Cho M, Chun JH, Cho MH, Park J et al. The poly-γ-d-glutamic acid capsule of Bacillus anthracis enhances lethal toxin activity. Infect Immun 2011;79:3846–3854 [CrossRef][PubMed]
    [Google Scholar]
  12. Leppla SH, Robbins JB, Schneerson R, Shiloach J. Development of an improved vaccine for anthrax. J Clin Invest 2002;110:141–144 [CrossRef][PubMed]
    [Google Scholar]
  13. Liu S, Moayeri M, Leppla SH. Anthrax lethal and edema toxins in anthrax pathogenesis. Trend Microbiol 2014;22:317–325[CrossRef]
    [Google Scholar]
  14. Sela-Abramovich S, Chitlaru T, Gat O, Grosfeld H, Cohen O et al. Novel and unique diagnostic biomarkers for Bacillus anthracis infection. Appl Environ Microbiol 2009;75:6157–6167 [CrossRef][PubMed]
    [Google Scholar]
  15. Shafferman A, Gat O, Ariel N, Theodor C, Haim G et al. Reverse vaccinology in Bacillus anthracis. In Shafferman A, Ordentlich A, Velan B. (editors) The Challenge of Highly Pathogenic Microorganisms: Mechanisms of Virulence and Novel Medical Countermeasures Dordrecht, The Netherlands: Springer; 2010; pp.295–306[CrossRef]
    [Google Scholar]
  16. Tran S-L, Guillemet E, Gohar M, Lereclus D, Ramarao N. CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase implicated in adhesion, biofilm formation, and virulence. J Bacteriol 2010;192:2638–2642 [CrossRef][PubMed]
    [Google Scholar]
  17. Kim SK, Shahid S, Kim SH, Park JH, Lee HT et al. Comparative analysis of virulence factors secreted by Bacillus anthracis Sterne at host body temperature. Lett Appl Microbiol 2012;54:306–312 [CrossRef][PubMed]
    [Google Scholar]
  18. Kim SK, Jung KH, Chai YG. Changes in Bacillus anthracis CodY regulation under host-specific environmental factor deprived conditions. BMC Genomics 2016;17:645 [CrossRef][PubMed]
    [Google Scholar]
  19. Kim SK, Jung KH, Yoon SN, Kim YK, Chai YG. Late-exponential gene expression in codY-deficient Bacillus anthracis in a host-like environment. Curr Microbiol 2016;73:714–720 [CrossRef][PubMed]
    [Google Scholar]
  20. Notredame C, Higgins DG, Heringa J. T-coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 2000;302:205–217 [CrossRef][PubMed]
    [Google Scholar]
  21. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Meth 2011;8:785–786[CrossRef]
    [Google Scholar]
  22. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F et al. CDD: NCBI's conserved domain database. Nucleic Acids Res 2015;43:D222–D226 [CrossRef][PubMed]
    [Google Scholar]
  23. Kim HU, Goepfert JM. A sporulation medium for Bacillus anthracis. J Appl Bacteriol 1974;37:265–267[PubMed][CrossRef]
    [Google Scholar]
  24. Ristroph JD, Ivins BE. Elaboration of Bacillus anthracis antigens in a new, defined culture medium. Infect Immun 1983;39:483–486[PubMed]
    [Google Scholar]
  25. Shatalin KY, Neyfakh AA. Efficient gene inactivation in Bacillus anthracis. FEMS Microbiol Lett 2005;245:315–319 [CrossRef][PubMed]
    [Google Scholar]
  26. Kim SH, Kim SK, Jung KH, Kim YK, Hwang HC et al. Proteomic analysis of the oxidative stress response induced by low-dose hydrogen peroxide in Bacillus anthracis. J Microbiol Biotechnol 2013;23:750–758[PubMed][CrossRef]
    [Google Scholar]
  27. Baik SY, Jung KH, Choi MR, Yang BH, Kim SH et al. Fluoxetine-induced up-regulation of 14-3-3zeta and tryptophan hydroxylase levels in RBL-2H3 cells. Neurosci Lett 2005;374:53–57 [CrossRef][PubMed]
    [Google Scholar]
  28. Drysdale M, Bourgogne A, Hilsenbeck SG, Koehler TM. atxA controls Bacillus anthracis capsule synthesis via acpA and a newly discovered regulator, acpB. J Bacteriol 2004;186:307–315[PubMed][CrossRef]
    [Google Scholar]
  29. Carr KA, Janes BK, Hanna PC. Role of the gerP operon in germination and outgrowth of Bacillus anthracis spores. PLoS One 2010;5:e9128 [CrossRef][PubMed]
    [Google Scholar]
  30. Pfeffer JM, Strating H, Weadge JT, Clarke AJ. Peptidoglycan O acetylation and autolysin profile of Enterococcus faecalis in the viable but nonculturable state. J Bacteriol 2006;188:902–908 [CrossRef][PubMed]
    [Google Scholar]
  31. Mavrici D, Marakalala MJ, Holton JM, Prigozhin DM, Gee CL et al. Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC. Proc Natl Acad Sci USA 2014;111:8037–8042 [CrossRef][PubMed]
    [Google Scholar]
  32. Hutchison EA, Miller DA, Angert ER. Sporulation in bacteria: beyond the standard model. Microbiol Spectr 2014;2: doi: 10.1128/microbiolspec. TBS-0013-2012 [CrossRef][PubMed]
    [Google Scholar]
  33. Rodrigues CDA, Marquis KA, Meisner J, Rudner DZ. Peptidoglycan hydrolysis is required for assembly and activity of the transenvelope secretion complex during sporulation in Bacillus subtilis. Mol Microbiol 2013;89:1039–1052 [CrossRef][PubMed]
    [Google Scholar]
  34. Moat AG, Foster JW, Spector MP. Microbial Physiology, 4 ed. New York: John Wiley; 2003
    [Google Scholar]
  35. Ohnishi R, Ishikawa S, Sekiguchi J. Peptidoglycan hydrolase LytF plays a role in cell separation with CwlF during vegetative growth of Bacillus subtilis. J Bacteriol 1999;181:3178–3184[PubMed]
    [Google Scholar]
  36. Ishikawa S, Hara Y, Ohnishi R, Sekiguchi J. Regulation of a new cell wall hydrolase gene, cwlF, which affects cell separation in Bacillus subtilis. J Bacteriol 1998;180:2549–2555[PubMed]
    [Google Scholar]
  37. Margot P, Wahlen M, Gholamhoseinian A, Piggot P, Karamata D et al. The lytE gene of Bacillus subtilis 168 encodes a cell wall hydrolase. J Bacteriol 1998;180:749–752[PubMed]
    [Google Scholar]
  38. Xu Q, Mengin-Lecreulx D, Liu XW, Patin D, Farr CL et al. Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains. MBio 2015;6:e02327-14 [CrossRef][PubMed]
    [Google Scholar]
  39. Bannantine JP, Lingle CK, Stabel JR, Ramyar KX, Garcia BL et al. MAP1272c encodes an NlpC/P60 protein, an antigen detected in cattle with Johne's Disease. Clin Vacc Immunol 2012;19:1083–1092[CrossRef]
    [Google Scholar]
  40. Bannantine JP, Lingle CK, Adam PR, Ramyar KX, Mcwhorter WJ et al. NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan. Protein Sci 2016;25:840–851 [CrossRef][PubMed]
    [Google Scholar]
  41. Ghuysen JM. Serine beta-lactamases and penicillin-binding proteins. Annu Rev Microbiol 1991;45:37–67 [CrossRef][PubMed]
    [Google Scholar]
  42. Imamura D, Kobayashi K, Sekiguchi J, Ogasawara N, Takeuchi M et al. spoIVH (ykvV), a requisite cortex formation gene, is expressed in both sporulating compartments of Bacillus subtilis. J Bacteriol 2004;186:5450–5459 [CrossRef][PubMed]
    [Google Scholar]
  43. Moriyama R, Fukuoka H, Miyata S, Kudoh S, Hattori A et al. Expression of a germination-specific amidase, SleB, of Bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores. J Bacteriol 1999;181:2373–2378[PubMed]
    [Google Scholar]
  44. Murray T, Popham DL, Setlow P. Identification and characterization of pbpC, the gene encoding Bacillus subtilis penicillin-binding protein 3. J Bacteriol 1996;178:6001–6005[PubMed][CrossRef]
    [Google Scholar]
  45. Pedersen LB, Murray T, Popham DL, Setlow P. Characterization of dacC, which encodes a new low-molecular-weight penicillin-binding protein in Bacillus subtilis. J Bacteriol 1998;180:4967–4973[PubMed]
    [Google Scholar]
  46. Yanouri A, Daniel RA, Errington J, Buchanan CE. Cloning and sequencing of the cell division gene pbpB, which encodes penicillin-binding protein 2B in Bacillus subtilis. J Bacteriol 1993;175:7604–7616[PubMed][CrossRef]
    [Google Scholar]
  47. Mcpherson DC, Popham DL. Peptidoglycan synthesis in the absence of class A penicillin-binding proteins in Bacillus subtilis. J Bacteriol 2003;185:1423–1431[PubMed][CrossRef]
    [Google Scholar]
  48. Wei Y, Havasy T, Mcpherson DC, Popham DL. Rod shape determination by the Bacillus subtilis class B penicillin-binding proteins encoded by pbpA and pbpH. J Bacteriol 2003;185:4717–4726[PubMed][CrossRef]
    [Google Scholar]
  49. Ragkousi K, Setlow P. Transglutaminase-mediated cross-linking of GerQ in the coats of Bacillus subtilis spores. J Bacteriol 2004;186:5567–5575 [CrossRef][PubMed]
    [Google Scholar]
  50. Zilhão R, Isticato R, Martins LO, Steil L, Völker U et al. Assembly and function of a spore coat-associated transglutaminase of Bacillus subtilis. J Bacteriol 2005;187:7753–7764 [CrossRef][PubMed]
    [Google Scholar]
  51. Fernandes CG, Plácido D, Lousa D, Brito JA, Isidro A et al. Structural and functional characterization of an ancient bacterial transglutaminase sheds light on the minimal requirements for protein cross-linking. Biochemistry 2015;54:5723–5734 [CrossRef][PubMed]
    [Google Scholar]
  52. Chitlaru T, Gat O, Gozlan Y, Ariel N, Shafferman A. Differential proteomic analysis of the Bacillus anthracis secretome: distinct plasmid and chromosome CO2-dependent cross talk mechanisms modulate extracellular proteolytic activities. J Bacteriol 2006;188:3551–3571 [CrossRef][PubMed]
    [Google Scholar]
  53. Chandramohan L, Ahn JS, Weaver KE, Bayles KW. An overlap between the control of programmed cell death in Bacillus anthracis and sporulation. J Bacteriol 2009;191:4103–4110 [CrossRef][PubMed]
    [Google Scholar]
  54. Lazar SW, Almirón M, Tormo A, Kolter R. Role of the Escherichia coli SurA protein in stationary-phase survival. J Bacteriol 1998;180:5704–5711[PubMed]
    [Google Scholar]
  55. Böth D, Schneider G, Schnell R. Peptidoglycan remodeling in Mycobacterium tuberculosis: comparison of structures and catalytic activities of RipA and RipB. J Mol Biol 2011;413:247–260 [CrossRef][PubMed]
    [Google Scholar]
  56. Ruggiero A, Marasco D, Squeglia F, Soldini S, Pedone E et al. Structure and functional regulation of RipA, a mycobacterial enzyme essential for daughter cell separation. Structure 2010;18:1184–1190 [CrossRef][PubMed]
    [Google Scholar]
  57. Ruggiero A, Squeglia F, Esposito C, Marasco D, Pedone E et al. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the resuscitation promoting factor interacting protein RipA from M. tuberculosis. Protein Pept Lett 2010;17:70–73[PubMed][CrossRef]
    [Google Scholar]
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