Mirolysin, a LysargiNase from Tannerella forsythia, proteolytically inactivates the human cathelicidin, LL-37.

Tannerella forsythia is a periodontal pathogen expressing six secretory proteolytic enzymes with a unique multidomain structure referred to as KLIKK proteases. Two of these proteases, karilysin and mirolysin, were previously shown to protect the bacterium against complement-mediated bactericidal activity. The latter metalloprotease, however, was not characterized at the protein level. Therefore, we purified recombinant mirolysin and subjected it to detailed biochemical characterization. Mirolysin was obtained as a 66 kDa zymogen, which autoproteolytically processed itself into a 31 kDa active form via truncations at both the N- and C-termini. Further autodegradation was prevented by calcium. Substrate specificity was determined by the S1' subsite of the substrate-binding pocket, which shows strong preference for Arg and Lys at the carbonyl side of a scissile peptide bond (P1' residue). The protease cleaved an array of host proteins, including human fibronectin, fibrinogen, complement proteins C3, C4, and C5, and the antimicrobial peptide, LL-37. Degradation of LL-37 abolished not only the bactericidal activity of the peptide, but also its ability to bind lipopolysaccharide (LPS), thus quenching the endotoxin proinflammatory activity. Taken together, these results indicate that, through cleavage of LL-37 and complement proteins, mirolysin might be involved in evasion of the host immune response.

Foci of endemic simian immunodeficiency virus infection in wild-living eastern chimpanzees (Pan troglodytes schweinfurthii).

Simian immunodeficiency virus of chimpanzees (SIVcpz) is the immediate precursor to human immunodeficiency virus type 1 (HIV-1), yet remarkably, the distribution and prevalence of SIVcpz in wild ape populations are unknown. Studies of SIVcpz infection rates in wild chimpanzees are complicated by the species' endangered status and by its geographic location in remote areas of sub-Saharan Africa. We have developed sensitive and specific urine and fecal tests for SIVcpz antibody and virion RNA (vRNA) detection and describe herein the first comprehensive prevalence study of SIVcpz infection in five wild Pan troglodytes schweinfurthii communities in east Africa. In Kibale National Park in Uganda, 31 (of 52) members of the Kanyawara community and 39 (of approximately 145) members of the Ngogo community were studied; none were found to be positive for SIVcpz infection. In Gombe National Park in Tanzania, 15 (of 20) members of the Mitumba community, 51 (of 55) members of the Kasekela community, and at least 10 (of approximately 20) members of the Kalande community were studied. Seven individuals were SIVcpz antibody and/or vRNA positive, and two others had indeterminate antibody results. Based on assay sensitivities and the numbers and types of specimens analyzed, we estimated the prevalence of SIVcpz infection to be 17% in Mitumba (95% confidence interval, 10 to 40%), 5% in Kasekela (95% confidence interval, 4 to 7%), and 30% in Kalande (95% confidence interval, 15 to 60%). For Gombe as a whole, the SIVcpz prevalence was estimated to be 13% (95% confidence interval, 7 to 25%). SIVcpz infection was confirmed in five chimpanzees by PCR amplification of partial pol and gp41/nef sequences which revealed a diverse group of viruses that formed a monophyletic lineage within the SIVcpzPts radiation. Although none of the 70 Kibale chimpanzees tested SIVcpz positive, we estimated the likelihood that a 10% or higher prevalence existed but went undetected because of sampling and assay limitations; this possibility was ruled out with 95% certainty. These results indicate that SIVcpz is unevenly distributed among P. t. schweinfurthii in east Africa, with foci or "hot spots" of SIVcpz endemicity in some communities and rare or absent infection in others. This situation contrasts with that for smaller monkey species, in which infection rates by related SIVs are generally much higher and more uniform among different groups and populations. The basis for the wide variability in SIVcpz infection rates in east African apes and the important question of SIVcpz prevalence in west central African chimpanzees (Pan troglodytes troglodytes) remain to be elucidated.

Amplification of a complete simian immunodeficiency virus genome from fecal RNA of a wild chimpanzee.

Current knowledge of the genetic diversity of simian immunodeficiency virus (SIVcpz) infection of wild chimpanzees (Pan troglodytes) is incomplete since few isolates, mostly from captive apes from Cameroon and Gabon, have been characterized; yet this information is critical for understanding the origins of human immunodeficiency virus type 1 (HIV-1) and the circumstances leading to the HIV-1 pandemic. Here, we report the first full-length SIVcpz sequence (TAN1) from a wild chimpanzee (Pan troglodytes schweinfurthii) from Gombe National Park (Tanzania), which was obtained noninvasively by amplification of virion RNA from fecal samples collected under field conditions. Using reverse transcription-PCR and a combination of generic and strain-specific primers, we amplified 13 subgenomic fragments which together spanned the entire TAN1 genome (9,326 bp). Distance and phylogenetic tree analyses identified TAN1 unambiguously as a member of the HIV-1/SIVcpz group of viruses but also revealed an extraordinary degree of divergence from all previously characterized SIVcpz and HIV-1 strains. In Gag, Pol, and Env proteins, TAN1 differed from west-central African SIVcpz and HIV-1 strains on average by 36, 30, and 51% of amino acid sequences, respectively, approaching distance values typically found for SIVs from different primate species. The closest relative was SIVcpzANT, also from a P. t. schweinfurthii ape, which differed by 30, 25, and 44%, respectively, in these same protein sequences but clustered with TAN1 in all major coding regions in a statistically highly significant manner. These data indicate that east African chimpanzees, like those from west-central Africa, are naturally infected by SIVcpz but that their viruses comprise a second, divergent SIVcpz lineage which appears to have evolved in relative isolation for an extended period of time. Our data also demonstrate that noninvasive molecular epidemiological studies of SIVcpz in wild chimpanzees are feasible and that such an approach may prove essential for unraveling the evolutionary history of SIVcpz/HIV-1 as well as that of other pathogens naturally infecting wild primate populations.