Insights into the Autoproteolytic Processing and Catalytic Mechanism of the Chlamydia trachomatis Virulence-Associated Protease CPAF.

CPAF (chlamydial protease-like activity factor) is a Chlamydia trachomatis protease that is translocated into the host cytosol during infection. CPAF activity results in dampened host inflammation signaling, cytoskeletal remodeling, and suppressed neutrophil activation. Although CPAF is an emerging antivirulence target, its catalytic mechanism has been unexplored to date. Steady state kinetic parameters were obtained for recombinant CPAF with vimentin-derived peptide substrates using a high-performance liquid chromatography-based discontinuous assay (kcat = 45 ± 0.6 s-1; kcat/Km = 0.37 ± 0.02 µM-1 s-1) or a new fluorescence-based continuous assay (kcat = 23 ± 0.7 s-1; kcat/Km = 0.29 ± 0.03 µM-1 s-1). Residues H105, S499, E558, and newly identified D103 were found to be indispensable for autoproteolytic processing by mutagenesis, while participation of C500 was ruled out despite its proximity to the S499 nucleophile. Pre-steady state kinetics indicated a burst kinetic profile, with fast acylation (kacyl = 110 ± 2 s-1) followed by slower, partially rate-limiting deacylation (kdeacyl = 57 ± 1 s-1). Both kcat- and kcat/Km-pH profiles showed single acidic limb ionizations with pKa values of 6.2 ± 0.1 and 6.5 ± 0.1, respectively. A forward solvent deuterium kinetic isotope effect of 2.6 ± 0.1 was observed for D2Okcatapp, but a unity effect was found for D2Okcat/Kmapp. The kcat proton inventory was linear, indicating transfer of a single proton in the rate-determining transition state, most likely from H105. Collectively, these data provide support for the classification of CPAF as a serine protease and provide a mechanistic foundation for the future design of inhibitors.

Diversity and Tropism of HIV-1 Rebound Virus Populations in Plasma Level After Treatment Discontinuation.

Human immunodeficiency virus-infected people discontinuing therapy experience a rebound in the virus level (hereafter, "rebound virus") from a persistent reservoir. We examined 10 samples from patients in AIDS Clinical Trials Group study A5068 with rebound virus, using single-genome amplification and Primer ID deep sequencing, to assess env genetic diversity of the virus population. Most rebound-virus populations showed significant diversity. All env examined required high levels of CD4 for entry, consistent with selection of replication in CD4(+) T cells. These results indicate that most people discontinuing therapy release a diverse population of virus and that this released virus has entry features of virus selected for replication in CD4(+) T cells, rather than in myeloid cells.

Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions.

UNLABELLED: HIV-1 requires the CD4 receptor and a coreceptor (CCR5 [R5 phenotype] or CXCR4 [X4 phenotype]) to enter cells. Coreceptor tropism can be assessed by either phenotypic or genotypic analysis, the latter using bioinformatics algorithms to predict tropism based on the env V3 sequence. We used the Primer ID sequencing strategy with the MiSeq sequencing platform to reveal the structure of viral populations in the V1/V2 and C2/V3 regions of the HIV-1 env gene in 30 late-stage and 6 early-stage subjects. We also used endpoint dilution PCR followed by cloning of env genes to create pseudotyped virus to explore the link between genotypic predictions and phenotypic assessment of coreceptor usage. We found out that the most stringently sequence-based calls of X4 variants (Geno2Pheno false-positive rate [FPR] of ≤2%) formed distinct lineages within the viral population, and these were detected in 24 of 30 late-stage samples (80%), which was significantly higher than what has been seen previously by using other approaches. Non-X4 lineages were not skewed toward lower FPR scores in X4-containing populations. Phenotypic assays showed that variants with an intermediate FPR (2 to 20%) could be either X4/dual-tropic or R5 variants, although the X4 variants made up only about 25% of the lineages with an FPR of <10%, and these variants carried a distinctive sequence change. Phylogenetic analysis of both the V1/V2 and C2/V3 regions showed evidence of recombination within but very little recombination between the X4 and R5 lineages, suggesting that these populations are genetically isolated. IMPORTANCE: Primer ID sequencing provides a novel approach to study genetic structures of viral populations. X4 variants may be more prevalent than previously reported when assessed by using next-generation sequencing (NGS) and with a greater depth of sampling than single-genome amplification (SGA). Phylogenetic analysis to identify lineages of sequences with intermediate FPR values may provide additional information for accurately predicting X4 variants by using V3 sequences. Limited recombination occurs between X4 and R5 lineages, suggesting that X4 and R5 variants are genetically isolated and may be replicating in different cell types or that X4/R5 recombinants have reduced fitness.

R5 Macrophage-Tropic HIV-1 in the Male Genital Tract.

The entry tropism of HIV-1 Env proteins from virus isolated from the blood and genital tract of five men with compartmentalized lineages was determined. The Env proteins isolated from the genital tract of subject C018 were macrophage-tropic proteins, while the remaining cloned env genes encoded R5 T cell-tropic proteins. The detection of a macrophage-tropic lineage of HIV-1 within the male genital tract strongly suggests that evolution of macrophage-tropic viruses can occur in anatomically isolated sites outside the central nervous system.

Compartmentalization, Viral Evolution, and Viral Latency of HIV in the CNS.

Human immunodeficiency virus type 1 (HIV-1) infection occurs throughout the body and can have dramatic physical effects, such as neurocognitive impairment in the central nervous system (CNS). Furthermore, examining the virus that resides in the CNS is challenging due to its location and can only be done using samples collected either at autopsy, indirectly form the cerebral spinal fluid (CSF), or through the use of animal models. The unique milieu of the CNS fosters viral compartmentalization as well as evolution of viral sequences, allowing for new cell types, such as macrophages and microglia, to be infected. Treatment must also cross the blood-brain barrier adding additional obstacles in eliminating viral populations in the CNS. These long-lived infected cell types and treatment barriers may affect functional cure strategies in people on highly active antiretroviral therapy (HAART).

Studies on the biosynthesis of the lipodepsipeptide antibiotic Ramoplanin A2.

Ramoplanin, a non-ribosomally synthesized peptide antibiotic, is highly effective against several drug-resistant Gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Recently, the biosynthetic cluster from the ramoplanin producer Actinoplanes ATCC 33076 was sequenced, revealing an unusual architecture of fatty acid and non-ribosomal peptide synthetase biosynthetic genes (NRPSs). The first steps towards understanding how these biosynthetic enzymes cooperatively interact to produce the depsipeptide product are expression and isolation of each enzyme to probe its specificity and function. Here we describe the successful production of soluble enzymes from within the ramoplanin locus and the confirmation of their specific role in biosynthesis. These methods may be broadly applicable to the production of biosynthetic enzymes from other natural product biosynthetic gene clusters, especially those that have been refractory to production in heterologous hosts despite standard expression optimization methods.

Chlamydia protease-like activity factor (CPAF): characterization of proteolysis activity in vitro and development of a nanomolar affinity CPAF zymogen-derived inhibitor.

During infection of epithelial cells, the obligate intracellular pathogen Chlamydia trachomatis secretes the serine protease Chlamydia protease-like activity factor (CPAF) into the host cytosol to regulate a range of host cellular processes through targeted proteolysis. Here we report the development of an in vitro assay for the enzyme and the discovery of a cell-permeable CPAF zymogen-based peptide inhibitor with nanomolar inhibitory affinity. Treating C. trachomatis-infected HeLa cells with this inhibitor prevented CPAF cleavage of the intermediate filament vimentin and led to the loss of vimentin cage surrounding the intracellular vacuole. Because Chlamydia is a genetically intractable organism, this inhibitor may serve as a tool for understanding the role of CPAF in pathogenesis.

The Chlamydia protease CPAF regulates host and bacterial proteins to maintain pathogen vacuole integrity and promote virulence.

The obligate intracellular bacterial pathogen Chlamydia trachomatis injects numerous effector proteins into the epithelial cell cytoplasm to manipulate host functions important for bacterial survival. In addition, the bacterium secretes a serine protease, chlamydial protease-like activity factor (CPAF). Although several CPAF targets are reported, the significance of CPAF-mediated proteolysis is unclear due to the lack of specific CPAF inhibitors and the diversity of host targets. We report that CPAF also targets chlamydial effectors secreted early during the establishment of the pathogen-containing vacuole ("inclusion"). We designed a cell-permeable CPAF-specific inhibitory peptide and used it to determine that CPAF prevents superinfection by degrading early Chlamydia effectors translocated during entry into a preinfected cell. Prolonged CPAF inhibition leads to loss of inclusion integrity and caspase-1-dependent death of infected epithelial cells. Thus, CPAF functions in niche protection, inclusion integrity and pathogen survival, making the development of CPAF-specific protease inhibitors an attractive antichlamydial therapeutic strategy.