The genome of Mycoplasma suis and its metabolism, pathogenesis and evolution

Ana Marcia de Sa Guimaraes, Purdue University

Abstract

Mycoplasma suis is an uncultivable, red blood cell (RBC) pathogen of pigs that causes hemolytic anemia or chronic, subclinical infections. In the studies reported herein, the genome of this organism was completely sequenced, analyzed and compared to other Mollicutes; and M.suis-specific molecular and serologic assays were developed to detect infected pigs. Accordingly, the first study describes the development of a highly sensitive and specific quantitative TaqMan® PCR (qPCR) assay that provided further insights into the blood bacterial load during acute and chronic disease. The assay was validated using samples from 80 sows and 3-6 months old pigs, a M. suis-infected, splenectomized pig (acute disease model; pig #1), and a naturally infected, non-splenectomized pig (chronic disease model, pig #2). The prevalence among field pigs was found to be 43.7% (35/80), with quantification values ranging from 6.93 x 10 ³ to 4.02 x 10⁹ organisms/mL of blood. An average of 4.86 x 10¹¹ organisms/mL of blood at peaks of bacteremia in pig #1 and 2.75 x 10⁸ organisms/mL of blood in pig #2 were detected throughout the study. The difference in these values explains our ability to visualize organisms attached to the RBCs on blood smears of acutely infected pigs at peaks of bacteremia, but not in chronically infected pigs. ^ The second study describes the whole genome sequencing and analyses of M. suis. It was observed that a great proportion of M. suis genes encodes for hypothetical proteins (61.3%), most of which are grouped into paralogous gene families (PGFs) (42.8%). We believe that some of these families are linked to antigenic variation, as observed in other mycoplasmas and blood-borne pathogens. We then used the predicted proteome to design a complete metabolic map of M. suis, which indicates metabolic reduction and evidence of adaption to the blood environment. The analysis of this map also suggests essential nutrients that may be needed in future attempts to cultivate M. suis in vitro. Interestingly, toxin orthologs were not identified. Thus, we propose that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs.^ The third study describes the identification of M. suis antigens and development of a microbead immunoassay (MIA) using Luminex® technology. A total of 6 antigens, including 4 proteins from PGFs, were identified; three of these proteins (GrpE, GAPN and one PGF protein) were used to develop the MIA. The MIA is more sensitive than the indirect hemagglutination (IHA) assay. We also observed a great number of qPCR-negative, MIA-positive animals when testing field samples, which suggests the possibility of infection clearance and/or low blood bacterial load that falls below the detection limit of our qPCR.^ The fourth study describes the comparative genomics and phylogenomics of the eight hemoplasmas sequenced to date. It was concluded that the hemoplasmas have highly dynamic genomes. This is supported by evidence of substantial gene gain and/or loss throughout evolution, loss of gene synteny, positional shuffling of genes from PGFs, and detection of horizontal gene transfer (HGT). They experienced metabolic reduction when compared to other Mollicutes, but PGFs are likely maintained and fixed within the bacterial population through positive selection. The hemoplasmas are using HGT as means of adapting to the blood environment by acquiring genes related to its metabolism. And finally, our phylogenetic analyses shows that hemoplasmas are not part of the pneumoniae group, as previously thought, and are the most divergent clade within the Mollicutes class.^ The fifth and final study briefly highlights possible mechanisms of antigenic variation of immunogenic PGFs. Phase variation through slipped-strand mispairing of homopolymeric guanine tracts and homologous DNA recombination are the suggested mechanisms employed by two of these PGFs.^ The studies reported herein greatly expanded our knowledge of the metabolism, pathogenesis and evolution of M. suis. These results will likely serve as basis for further research related to in vitro cultivation systems, M. suis interaction with RBCs, mechanisms of immune system evasion and diagnostics.^

Degree

Ph.D.

Advisors

Joanne B. Messick, Purdue University.

Subject Area

Biology, Microbiology|Biology, Veterinary Science

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