Expression and contribution to virulence of each polysaccharide capsule of Bacillus cereus strain G9241

Jennifer M. Scarff, Yuliya I. Seldina, James M. Vergis, Christy L. Ventura, Alison D. O'Brien, Theresa M. Koehler
2018 PLoS ONE  
Bacillus cereus strain G9241 was isolated from a patient with pneumonia who had an anthrax-like illness. Like Bacillus anthracis, the virulence of G9241 is dependent on two large plasmids. In G9241 those plasmids are pBCXO1 and pBC210. There is a multi-gene capsule locus on each of these virulence plasmids, and both capsules are produced by G9241 in vitro and in mice. The hasACB operon on pBCXO1 is responsible for production of a hyaluronic acid (HA) capsule. The locus on pBC210 encodes a
more » ... 210 encodes a putative tetrasaccharide (TS) capsule that assembles in a Wzy-dependent manner. We found that the pBC210 capsule locus is transcribed as two operons and identified the promoter regions responsible for transcription. We constructed isogenic mutants to assess the role of genes in the two TS capsule operons in production of the capsule. Spores of strains deficient in production of either the HA or TS capsule were inoculated subcutaneously or intranasally into A/J and C57BL/6 mice to determine the lethal dose 50% of each bacterial mutant by each route of infection. The loss of the HA capsule attenuated G9241 more than the loss of the TS capsule for both infection routes in both mouse strains. Overall, our data further characterize the unique TS capsule on pBC210 and demonstrate that the two capsules do not have the same impact on virulence of G9241. OPEN ACCESS Citation: Scarff JM, Seldina YI, Vergis JM, Ventura CL, O'Brien AD (2018) Expression and contribution to virulence of each polysaccharide capsule of Bacillus cereus strain G9241. PLoS ONE 13(8): e0202701. https://doi.org/10.to be construed as official similar to pXO1 and also encodes PA, LF, EF, and AtxA1 while the other plasmid, pBC210 (formerly pBC218), is unique compared to pXO2 [7] . In fact, pBC210 encodes a PA homolog (PA2), a novel ADP-ribosylating toxin, certhrax, and an AtxA homolog, AtxA2 [7-10]. Each virulence plasmid also contains genetic loci that encode the proteins to construct a polysaccharide capsule. Plasmid pBCXO1 carries the hasACB operon that is responsible for production of a hyaluronic acid (HA) capsule, while pBC210 contains a capsule locus that is required for elaboration of a putative tetrasaccharide (TS) capsule [7] . The two capsules in G9241 are both produced in vivo [11, 12] . The HA capsule is regulated by AtxA1, and the TS capsule is regulated by either AtxA1 or AtxA2 [12] . The hasACB operon on pBCXO1 is similar to the hasABC operons in Streptococcus pyogenes strains and is responsible for the production of an HA capsule, a disaccharide of glucuronic acid and N-acteylglucosamine. The HasA synthase assembles the high molecular weight repeating disaccharide polymers, and HasC and HasB are involved in synthesis of the nucleotide sugar precursors [13] . The capsule locus on pBC210 was initially characterized as a fifteen gene locus responsible for production of the TS capsule [7] . The final nine genes in the capsule locus were later identified as bpsX-H [11] . The TS capsule locus is similar in content to the Wzy-dependent polysaccharide capsules of Streptococcus pneumoniae [14] . The Wzy-dependent S. pneumoniae capsule loci encode four regulatory proteins, a glycosyltransferase for each sugar in the subunit, a flippase, a polymerase, and enzymes required to synthesize sugars that are unique to the capsule. The regulatory proteins are conserved among all serotypes, and the other proteins are serotype-specific [14, 15] . The synthesis of these types of capsules is initiated by the successive addition of each sugar subunit to a carrier molecule on the interior surface of the bacterial membrane. The Wzx flippase then "flips" the sugar moiety to the outer surface of the membrane and Wzy polymerase transfers the already formed chains on the surface to the recently "flipped" subunit. Other B. cereus strains have emerged in the Southeastern United States that caused a severe anthrax-like respiratory illness in metalworkers or a cutaneous infection in a healthy adult man [16] [17] [18] [19] [20] . These strains are encapsulated but do not contain pXO2-like plasmids or the PDGA capsule locus [18, 21, 22] . However, the strains do contain pBCXO1-like plasmids that encode an HA capsule in addition to the PA, LF, and EF associated with B. anthracis [19, 21, 23] . Only about half of the isolated strains have been either PCR-or sequence-confirmed to have pBC210 or the TS capsule locus [19, 21, 22] . Although one strain, BcFL2013, isolated from the cutaneous infection, has a partial plasmid of pBC210 that does not include the TS capsule locus [23] . In addition to these emerging U.S. strains, isolates of B. cereus biovar anthracis have been found as the sources of fatal infections in great apes in Cameroon and Côte d'Ivoire and wild and domestic animals in the Central African Republic and the Democratic Republic of the Congo [24, 25] . Only the strains from the great apes in Cameroon and Côte d'Ivoire have been further genetically characterized. These strains harbor both pBCXO1 and a pXO2-like plasmid, pBCXO2. Consequently, these strains produce PA, LF, and EF as well as the HA and PDGA capsules [26, 27] . In this study, we used G9241 as a prototypical U.S. emergent strain of B. cereus with B. anthracis-associated virulence factors. We determined that the unique pBC210 TS capsule locus is comprised of two operons and identified transcriptional start sites and promoter regions for each operon. We also demonstrated that deletion of a glycosyltransferase or the polymerase from the TS capsule locus abrogated production of the capsule. Lastly, we compared the virulence of G9241 and two isogenic mutants that produced only HA or TS capsules Two polysaccharide capsules of G9241
doi:10.1371/journal.pone.0202701 pmid:30133532 fatcat:p6uvqxuljvhdzc4sgrzwrj6x6i