Structural and Evolutionary Analyses Show Unique Stabilization Strategies in the Type IV Pili of Clostridium difficile

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, biofilm formation, cellular adhesion and horizontal gene transfer. However, many Gram-positive species, including C. difficile, also produce Type IV pili. Here, we identify the major subunit of the Type IV pili of C. difficile, PilA1, and describe multiple three-dimensional structures of PilA1, demonstrating the diversity found in three strains of C. difficile. We also model the incorporation of both PilA1 and a minor pilin, PilJ, into the pilus fiber. Although PilA1 contains no cysteine residues, and therefore cannot form the disulfide bonds found in all Gram-negative Type IV pilins, it adopts unique strategies to achieve a typical pilin fold. The structures of PilA1 and PilJ exhibit similarities with the Type IVb pilins from Gram-negative bacteria that suggest that the Type IV pili of C. difficile are involved in microcolony formation

Structure of \u3ci\u3eClostridium difficile\u3c/i\u3e PilJ Exhibits Unprecedented Divergence from Known Type IV Pilins

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Grampositive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C. difficile may assemble and function. PilJ has several unique structural features, including a dual-pilin fold and the incorporation of a structural zinc ion. We show that PilJ is incorporated into Type IV pili in C. difficile and present a model in which the incorporation of PilJ into pili exposes the C-terminal domain of PilJ to create a novel interaction surface

Lack of Detectable HIV-1 Molecular Evolution during Suppressive Antiretroviral Therapy

A better understanding of changes in HIV-1 population genetics with combination antiretroviral therapy (cART) is critical for designing eradication strategies. We therefore analyzed HIV-1 genetic variation and divergence in patients' plasma before cART, during suppression on cART, and after viral rebound. Single-genome sequences of plasma HIV-1 RNA were obtained from HIV-1 infected patients prior to cART (N = 14), during suppression on cART (N = 14) and/or after viral rebound following interruption of cART (N = 5). Intra-patient population diversity was measured by average pairwise difference (APD). Population structure was assessed by phylogenetic analyses and a test for panmixia. Measurements of intra-population diversity revealed no significant loss of overall genetic variation in patients treated for up to 15 years with cART. A test for panmixia, however, showed significant changes in population structure in 2/10 patients after short-term cART (<1 year) and in 7/10 patients after long-term cART (1-15 years). The changes consisted of diverse sets of viral variants prior to cART shifting to populations containing one or more genetically uniform subpopulations during cART. Despite these significant changes in population structure, rebound virus after long-term cART had little divergence from pretherapy virus, implicating long-lived cells infected before cART as the source for rebound virus. The appearance of genetically uniform virus populations and the lack of divergence after prolonged cART and cART interruption provide strong evidence that HIV-1 persists in long-lived cells infected before cART was initiated, that some of these infected cells may be capable of proliferation, and that on-going cycles of viral replication are not evident

ART Suppresses Plasma HIV-1 RNA to a Stable Set Point Predicted by Pretherapy Viremia

Current antiretroviral therapy is effective in suppressing but not eliminating HIV-1 infection. Understanding the source of viral persistence is essential for developing strategies to eradicate HIV-1 infection. We therefore investigated the level of plasma HIV-1 RNA in patients with viremia suppressed to less than 50–75 copies/ml on standard protease inhibitor- or non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy using a new, real-time PCR-based assay for HIV-1 RNA with a limit of detection of one copy of HIV-1 RNA. Single copy assay results revealed that >80% of patients on initial antiretroviral therapy for 60 wk had persistent viremia of one copy/ml or more with an overall median of 3.1 copies/ml. The level of viremia correlated with pretherapy plasma HIV-1 RNA but not with the specific treatment regimen. Longitudinal studies revealed no significant decline in the level of viremia between 60 and 110 wk of suppressive antiretroviral therapy. These data suggest that the persistent viremia on current antiretroviral therapy is derived, at least in part, from long-lived cells that are infected prior to initiation of therapy

Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples

The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick), we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples (N = 134) or prostate specimens (N = 19) from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer

A 28-Year History of HIV-1 Drug Resistance and Transmission in Washington, DC

Washington, DC consistently has one of the highest annual rates of new HIV-1 diagnoses in the United States over the last 10 years. To guide intervention and prevention strategies to combat DC HIV infection, it is helpful to understand HIV transmission dynamics in a historical context. Toward this aim, we conducted a retrospective study (years 1987–2015) of 3,349 HIV pol sequences (1,026 bp) from 1,995 individuals living in the DC area belonging to three different cohorts. We coupled HIV sequence data with clinical information (sex, risk factor, race/ethnicity, viral load, subtype, anti-retroviral regimen) to identify circulating drug resistant mutations (DRM) and transmission clusters and assess their persistence over time. Of the transmission clusters identified in the DC area, 78.0 and 31.7% involved MSM and heterosexuals, respectively. The longest spread of time for a single cluster was 5 years (2007–2012) using a distance-based network inference approach and 27 years (1987–2014) using a maximum likelihood phylogenetic approach. We found eight subtypes and nine recombinants. Genetic diversity increased steadily over time with a slight peak in 2009 and remained constant thereafter until 2015. Nucleotide diversity also increased over time while relative genetic diversity (BEAST) remained relatively steady over the last 28 years with slight increases since 2000 in subtypes B and C. Sequences from individuals on drug therapy contained the highest total number of DRMs (1,104–1,600) and unique DRMs (63–97) and the highest proportion (&gt;20%) of resistant individuals. Heterosexuals (43.94%), MSM (40.13%), and unknown (44.26%) risk factors showed similar prevalence of DRMs, while injection drug users had a lower prevalence (33.33%). Finally, there was a 60% spike in the number of codons with DRMs between 2007 and 2010. Past patterns of HIV transmission and DRM accumulation over time described here will help to predict future efficacy of ART drugs based on DRMs persisting over time and identify risk groups of interest for prevention and intervention efforts within the DC population. Our results show how longitudinal data can help to understand the temporal dynamics of HIV-1 at the local level

Human Herpesvirus Replication and Abnormal CD8+ T Cell Activation and Low CD4+ T Cell Counts in Antiretroviral-Suppressed HIV-Infected Patients

Most HIV-infected patients receiving virologically suppressive antiretroviral therapy continue to have abnormal, generalized T cell activation. We explored whether the degree of ongoing cytomegalovirus (CMV), Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) replication was associated with higher virus-specific T cell activation and the failure to achieve normal absolute CD4+ T cell counts in the face of long-term suppressive antiretroviral therapy.Longitudinally collected PBMC and saliva specimens obtained from HIV-infected patients on effective antiretroviral therapy for at least one year (plasma HIV RNA <75 copies/mL) were examined using a multiplex CMV, EBV and KSHV DNA PCR assay. Eleven cases were chosen who had CD8+ T cell CD38+HLA-DR+ expression >10% and plateau absolute CD4+ T cell counts <500 cells/microL. Five controls from the same study had CD8+ T cell CD38 expression <10% and plateau absolute CD4+ T cell counts >500 cells/microL.Among all subjects combined, 18% of PMBC samples were positive for CMV DNA, and 27%, 73% and 24% of saliva samples were positive for CMV, EBV and KSHV DNA, respectively. No significant differences or trends were observed between cases and controls in proportions of all CMV, EBV or KSHV DNA positive specimens, proportions of subjects in each group that intermittently or continuously shed CMV, EBV or KSHV DNA in saliva, or the median number of genome copies of CMV, EBV and KSHV DNA in saliva. Overall, number of genome copies in saliva were lower for KSHV than for CMV and lower for CMV than for EBV. Although replication of CMV, EBV and KSHV persists in many antiretroviral-suppressed, HIV-infected patients, we observed no evidence in this pilot case-control study that the magnitude of such human herpesvirus replication is associated with abnormally increased CD8+ T cell activation and sub-normal plateau absolute CD4+ T cell counts following virologically suppressive antiretroviral therapy

Immune control of HIV-1 infection after therapy interruption: immediate versus deferred antiretroviral therapy

Abstract Background The optimal stage for initiating antiretroviral therapies in HIV-1 bearing patients is still a matter of debate. Methods We present computer simulations of HIV-1 infection aimed at identifying the pro et contra of immediate as compared to deferred Highly Active Antiretroviral Therapy (HAART). Results Our simulations highlight that a prompt specific CD8+ cytotoxic T lymphocytes response is detected when therapy is delayed. Compared to very early initiation of HAART, in deferred treated patients CD8+ T cells manage to mediate the decline of viremia in a shorter time and, at interruption of therapy, the virus experiences a stronger immune pressure. We also observe, however, that the immunological effects of the therapy fade with time in both therapeutic regimens. Thus, within one year from discontinuation, viral burden recovers to the value at which it would level off in the absence of therapy. In summary, simulations show that immediate therapy does not prolong the disease-free period and does not confer a survival benefit when compared to treatment started during the chronic infection phase. Conclusion Our conclusion is that, since there is no therapy to date that guarantees life-long protection, deferral of therapy should be preferred in order to minimize the risk of adverse effects, the occurrence of drug resistances and the costs of treatment.</p

The Effect of Raltegravir Intensification on Low-level Residual Viremia in HIV-Infected Patients on Antiretroviral Therapy: A Randomized Controlled Trial

In a double-blind trial, Rajesh Gandhi and colleagues detect no significant reduction in viral load after people with low-level HIV viremia added an integrase inhibitor to their treatment regimen