GapA and CrmA Coexpression Is Essential for Mycoplasma gallisepticum Cytadherence and Virulence

Abstract

It was previously demonstrated that avirulent Mycoplasma gallisepticum strain R high (passage 164) is lacking three proteins that are expressed in its virulent progenitor, strain R low (passage 15). These proteins were identified as the cytadhesin molecule GapA, the putative cytadhesin-related molecule CrmA, and a component of a high-affinity transporter system, HatA. Complementation of R high with wild-type gapA restored expression in the transformant (GT5) but did not restore the cytadherence phenotype and maintained avirulence in chickens. These results suggested that CrmA might play an essential role in the M. gallisepticum cytadherence process. CrmA is encoded by the second gene in the gapA operon and shares significant sequence homology to the ORF6 gene of Mycoplasma pneumoniae, which has been shown to play an accessory role in the cytadherence process. Complementation of R high with wild-type crmA resulted in the transformant (SDCA) that lacked the cytadherence and virulence phenotype comparable to that found in R high and GT5. In contrast, complementation of R high with the entire wild-type gapA operon resulted in the transformant (GCA1) that restored cytadherence to the level found in wild-type R low . In vivo pathogenesis trials revealed that GCA1 had regained virulence, causing airsacculitis in chickens. These results demonstrate that both GapA and CrmA are required for M. gallisepticum cytadherence and pathogenesis.Mycoplasma gallisepticum is one infectious agent initiating the chronic respiratory disease complex in chickens and is the primary agent of infectious sinusitis in turkeys (74). This bacterium has developed a wide array of surface molecules that are involved in cytadherence to host cells (9,17,22,51). GapA is considered the primary cytadhesin. Goh et al. (21) identified its gene in M. gallisepticum based on its nucleotide sequence homology to the Mycoplasma pneumoniae cytadhesin ADP1 gene. Subsequent studies showed that anti-GapA Fab fragments were able to significantly inhibit M. gallisepticum cytadherence (22). Troy (69) reported that GapA was absent in avirulent M. gallisepticum. These data led us to hypothesize that complementation of GapA expression in strain R high via Tn4001 might restore cytadherence and perhaps virulence. Neither cytadherence nor virulence was restored upon the gapA-complemented strain R high , transformant GT5 (56, 57). This indicated that other factors might play an important role in M. gallisepticum cytadherence. Protein profile comparison between virulent and avirulent strains showed that, in addition to GapA, two other proteins are absent in R high (69). One of these proteins was found to be encoded by the second gene of the gapA operon. Its gene product shows significant sequence homology with the precursor of M. pneumoniae ORF6 gene products, which are known to play accessory roles in P1 (ADP1)-mediated cytadherence (35,36,40,66 The aim of this study was to evaluate the role of CrmA in M. gallisepticum cytadherence and virulence. We demonstrate that coexpr...

GapA+ Mycoplasma gallisepticum ts-11 has improved vaccine characteristics

“…The crmA gene is located downstream of the gapA gene, as part of the same operon (Papazisi et al, 2000). Both GapA and CrmA are expressed in virulent M. gallisepticum strain R-low (Rodriguez & Kleven, 1980), and co-expression of GapA and CrmA is essential for cytadherence and virulence in M. gallisepticum (Papazisi et al, 2002). The absence of GapA, but not CrmA, has been observed in the ts-11 vaccine strain (Mudahi-Orenstein et al, 2003).…”

Attachment organelle ultrastructure correlates with phylogeny, not gliding motility properties, in Mycoplasma pneumoniae relatives

“…Mean speeds reported in this cluster range from 50 nm s 21 for M. amphoriforme to .300 nm s 21 for M. pneumoniae (Hatchel et al, 2006). Homologues of the M. pneumoniae attachment organelle proteins required for cytadherence and motility are restricted to the M. pneumoniae cluster (Tham et al, 1994;Dhandayuthapani et al, 1999;Papazisi et al, 2002), making it likely that a common fundamental mechanism involving this set of proteins underlies attachment organelle-mediated functions in each of these organisms. For most of these species, detailed analysis of the dimensions of the electron-dense cores has not been performed.…”

Development of a replicable oriC plasmid for Mycoplasma gallisepticum and Mycoplasma imitans, and gene disruption through homologous recombination in M. gallisepticum

“…Recently the genome of M. gallisepticum was sequenced completely (Papazisi et al, 2003), allowing prediction of the function of specific genes. However, genetic studies of M. gallisepticum have been limited by the lack of genetic tools for its manipulation, with most studies using transposons Tn916 (Dybvig & Cassell, 1987;Dybvig & Alderete, 1988;Whetzel et al, 2003) and Tn4001 (Bearson et al, 2003;Hedreyda et al, 1993;Hudson et al, 2006;Mahairas & Minion, 1989;Mudahi-Orenstein et al, 2003;Papazisi et al, 2002;Whetzel et al, 2003;Winner et al, 2003) or suicide vectors to study gene function. However, the random insertion of the transposon in the genome of the organism does not allow specific targeting of a gene of interest, and random integration can confound analyses of gene expression (Dybvig & Cassell, 1987;Mahairas & Minion, 1989).…”