Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum

Abstract

BackgroundAnaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain.ResultsAlthough in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S.ConclusionsOur study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.

Infection of Ixodes spp. tick cells with different Anaplasma phagocytophilum isolates induces the inhibition of apoptotic cell death

“…As expected, the results showed that the sheep isolate clustered together with Norway variant2 isolate obtained from a lamb. The L610 dog isolate clustered together with isolates from horses, ticks, humans, rodents and dogs but was different from the previously reported Dog2 isolate (Al-Khedery et al, 2012). Finally, as expected, the human NY18 isolate clustered together with other human isolates.…”

“…For comparison, the same sequences were obtained for 9 isolates of which the genomes have been sequenced and are available in Genbank. The isolates CRT35 (NCBI reference sequence JFBI01000001; Massung et al, 2007;Al-Khedery et al, 2012) and CRT38 (NCBI reference sequence NZ_APHI00000000) were isolated from I. scapularis ticks, the isolate Dog2 was isolated from a dog (NCBI reference sequence CP006618; Al-Khedery et al, 2012), the isolate JM was obtained from a rodent (NCBI reference sequence CP006617; Al-Khedery et al, 2012), the MRK isolate was obtained from a horse (NCBI reference sequence JFBH00000000; Al-Khedery et al, 2012), the (Katoh and Standley, 2013). After alignment, regions with gaps were removed and 2354 gap-free sites were used in maximum likelihood (ML) phylogenetic analysis as implemented in PhyML (v3.0 aLRT) (Anisimova and Gascuel, 2006;Guindon and Gascuel, 2003).…”

Secretome of obligate intracellularRickettsia

“…8A ). Interestingly, other Rickettsiales genomes encode duplicate ( Orientia , Neorickettsia , Midichloria ) or proliferated ( Wolbachia , Anaplasma , and Ehrlichia ) rvhB2 genes (Nelson et al ., 2008, Gillespie et al ., 2010, Sutten et al ., 2010, Al-Khedery et al ., 2012). Some proliferated RvhB2 genes have become co-opted into a surface antigen family, possibly to cope for the lack of LPS biosynthesis (Gillespie et al ., 2010) .…”

“…Many RvhB6 proteins across Rickettsiales have repeat regions within the N- and/or C-terminal extensions, and a staggering degree of sequence length and variation across strains of A . phagocytophilum has been reported within a repeat region of one RvhB6 protein (Al-Khedery et al ., 2012) . Remarkably, all four rvhB6 genes of E .…”

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

“…We expanded our studies by testing the functionality of chimeric T4CPs composed of TraJ's NTD joined to the soluble domains of VirD4 Ap and VirD4 Wp from A. phagocytophilum and W. pipientis, respectively. Rickettsial species carry T4SS gene clusters, and a growing body of evidence indicates that type IV secretion is a general requirement for intracellular survival and proliferation (10,43,65,66). Identification of T4SS effectors, however, has been hampered by the inability to grow rickettsial species axenically and by lack of robust genetic systems.…”