Chaperone proteins are redundant in nature and, to achieve their function, they bind a large repertoire of client proteins. DnaK is a bacterial chaperone protein that recognizes misfolded and aggregated proteins and drives their folding and intracellular trafficking. Some Mycoplasmas are associated with cancers, and we demonstrated that infection with a strain of Mycoplasma fermentans isolated in our lab promoted lymphoma in a mouse model. Its DnaK is expressed intracellularly in infected cells, it interacts with key proteins to hamper essential pathways related to DNA repair and p53 functions and uninfected cells can take-up extracellular DnaK. We profile here for the first time the eukaryotic proteins interacting with DnaK transiently expressed in five cancer cell lines. A total of 520 eukaryotic proteins were isolated by immunoprecipitation and identified by Liquid Chromatography Mass Spectrometry (LC-MS) analysis. Among the cellular DnaK-binding partners, 49 were shared between the five analyzed cell lines, corroborating the specificity of the interaction of DnaK with these proteins. Enrichment analysis revealed multiple RNA biological processes, DNA repair, chromatin remodeling, DNA conformational changes, protein-DNA complex subunit organization, telomere organization and cell cycle as the most significant ontology terms. This is the first study to show that a bacterial chaperone protein interacts with key eukaryotic components thus suggesting DnaK could become a perturbing hub for the functions of important cellular pathways. Given the close interactions between bacteria and host cells in the local microenvironment, these results provide a foundation for future mechanistic studies on how bacteria interfere with essential cellular processes.

Several species of mycoplasmas, including Mycoplasma fermentans, are associated with certain human cancers. We previously isolated and characterized in our laboratory a strain of human mycoplasma M. fermentans subtype incognitus (MF-I1) able to induce lymphoma in a Severe Combined Immuno-Deficient (SCID) mouse model, and we demonstrated that its chaperone protein, DnaK, binds and reduces functions of human poly-ADP ribose polymerase-1 (PARP1) and ubiquitin carboxyl-terminal hydrolase protein-10 (USP10), which are required for efficient DNA repair and proper p53 activities, respectively. We also showed that other bacteria associated with human cancers (including Mycoplasmapneumoniae, Helicobacterpylori, Fusobacteriumnucleatum, Chlamydiathrachomatis, and Chlamydia pneumoniae) have closely related DnaK proteins, indicating a potential common mechanism of cellular transformation. Here, we quantify dnaK mRNA copy number by RT-qPCR analysis in different cellular compartments following intracellular MF-I1 infection of HCT116 human colon carcinoma cells. DnaK protein expression in infected cells was also detected and quantified by Western blot. The amount of viable intracellular mycoplasma reached a steady state after an initial phase of growth and was mostly localized in the cytoplasm of the invaded cells, while we detected a logarithmically increased number of viable extracellular bacteria. Our data indicate that, after invasion, MF-I1 is able to establish a chronic intracellular infection. Extracellular replication was more efficient while MF-I1 cultured in cell-free axenic medium showed a markedly reduced growth rate. We also identified modifications of important regulatory regions and heterogeneous lengths of dnaK mRNA transcripts isolated from intracellular and extracellular MF-I1. Both characteristics were less evident in dnaK mRNA transcripts isolated from MF-I1 grown in cell-free axenic media. Taken together, our data indicate that MF-I1, after establishing a chronic infection in eukaryotic cells, accumulates different forms of dnaK with efficient RNA turnover.

Studies of the human microbiome have elucidated an array of complex interactions between prokaryotes and their hosts. However, precise bacterial pathogen–cancer relationships remain largely elusive, although several bacteria, particularly those establishing persistent intra-cellular infections, like mycoplasmas, can alter host cell cycles, affect apoptotic pathways, and stimulate the production of inflammatory substances linked to DNA damage, thus potentially promoting abnormal cell growth and transformation. Consistent with this idea, in vivo experiments in several chemically induced or genetically deficient mouse models showed that germ-free conditions reduce colonic tumor formation. We demonstrate that mycoplasma DnaK, a chaperone protein belonging to the Heath shock protein (Hsp)-70 family, binds Poly-(ADP-ribose) Polymerase (PARP)-1, a protein that plays a critical role in the pathways involved in recognition of DNA damage and repair, and reduces its catalytic activity. It also binds USP10, a key p53 regulator, reducing p53 stability and anti-cancer functions. Finally, we showed that bystander, uninfected cells take up exogenous DnaK—suggesting a possible paracrine function in promoting cellular transformation, over and above direct mycoplasma infection. We propose that mycoplasmas, and perhaps certain other bacteria with closely related DnaK, may have oncogenic activity, mediated through the inhibition of DNA repair and p53 functions, and may be involved in the initiation of some cancers but not necessarily involved nor necessarily even be present in later stages.

Background and aims Mycoplasmas are ubiquitous pathogens found not only in humans but also in animals, plants, insects and soil. Though they usually grow better in an aerobic environment, mycoplasmas are also facultative anaerobic microorganisms. Following infection, the transition of a microorganism from a normal environment into an anaerobic one (e.g. dead or dying tissue) may result in production of a higher number of bacterial toxins. The resolution of the bacterial proteome during the aerobic/anaerobic switch could thus allow the identification of potential pathogenic determinants and pathways. Methods We used two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight/tandem mass spectroscopy (MALDI-TOF MS/MS) and subsequent mass spectrometric analysis to characterize the liposoluble and hydrosoluble protein fractions of a strain of Mycoplasma fermentans isolated in our lab (MFI), that was cultured under either aerobic or anaerobic conditions. Results We identified the 27 most abundant proteins in the liposoluble fraction and the 30 most abundant proteins in the hydrosoluble fraction and determined their modulation under aerobic and anaerobic growth. By using Protein ANalysis TrougH Evolutionary Relationships (PANTHER) and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) software analysis tools, we were able to identify, define and organize the function of each protein, as well as to determine the specific interactome. Conclusions Our work provides the first proteome reference map of Mycoplasma fermentan s obtained under aerobic and anaerobic growing conditions. These data may help to better understand the mechanisms of pathogenicity of this microorganism and define new diagnostic targets.

We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53(-/-) SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.

Hydrogen sulfide (H2S), previously only considered a toxic environmental air pollutant, is now increasingly recognized as an important signaling molecule able to modulate several cellular pathways in many human tissues. As demonstrated in recent studies, H2S is produced endogenously in response to different cellular stimuli and plays different roles in controlling a number of physiological responses. The precise role of H2S in inflammation is still largely unknown. In particular, the role of H2S in the regulation of the inflammatory response in acute and chronic infections is being actively investigated because of its potential therapeutic use. To study the effect of H2S as an anti-inflammatory mediator during bacterial infections, we developed an ex vivo model of primary cells and cell lines infected with Mycoplasma. Our data demonstrate a dichotomic effect of H2S on the NF-kB and Nrf-2 molecular pathways, which were inhibited and stimulated, respectively.

The common gamma molecule (gamma c) is a shared signaling receptor subunit used by six gamma c-cytokines. These cytokines play crucial roles in the differentiation of the mature immune system and are involved in many human diseases. Moreover, recent studies suggest that multiple gamma c-cytokines are pathogenically involved in a single disease, thus making the shared gamma c-molecule a logical target for therapeutic intervention. However, the current therapeutic strategies seem to lack options to treat such cases, partly because of the lack of appropriate neutralizing antibodies recognizing the gamma c and, more importantly, because of the inherent and practical limitations in the use of monoclonal antibodies. By targeting the binding interface of the gamma c and cytokines, we successfully designed peptides that not only inhibit multiple gamma c-cytokines but with a selectable target spectrum. Notably, the lead peptide inhibited three gamma c-cytokines without affecting the other three or non-gamma c-cytokines. Biological and mutational analyses of our peptide provide new insights to our current understanding on the structural aspect of the binding of gamma c-cytokines the gamma c-molecule. Furthermore, we provide evidence that our peptide, when conjugated to polyethylene glycol to gain stability in vivo, efficiently blocks the action of one of the target cytokines in animal models. Collectively, our technology can be expanded to target various combinations of gamma c-cytokines and thereby will provide a novel strategy to the current anti-cytokine therapies against immune, inflammatory, and malignant diseases.

HTLV-1 is the first retrovirus that has been connected to human cancer. Since its discovery in 1978 by the Gallo group, research has been focused on the elucidation of the mechanism by which the virus transforms human T cells and causes a fatal T-cell leukemia called adult-T cell leukemia (ATL). Despite 30 years of research, ATL still remains a challenge to cure. A series of recent studies have identified a novel and important element of the HTLV-1 that is coded by the anti-sense strand of the HTLV-1. This factor, named HBZ (HTLV-1 basic leucine-zipper factor), has been demonstrated to cause a T-cell leukemia in a transgenic mouse model (Satou et al. 2011). In addition, HBZ functions in 2 forms, as a regulatory RNA and as a nuclear protein. We asked which of the 2 forms of HBZ is involved in T cell transformation. We generated a humanized mouse model by transducing HBZ components (i.e., RNA only, or Protein only, or both) to human CD34+ progenitor cells and adoptively transferred them into immune-compromised SCID-NSG mice. The results suggest that both RNA and protein of HBZ are needed to cause malignancy in mice. We then investigated the roles of individual components in the cellular transformation, and observed that HBZ-RNA compromises cellular safe-guard mechanism against chromosomal damages and protects mutated cells from death. Surprisingly, the p53 pathway did function in HBZ-RNA positive cells, but it has been disconnected from the death-inducing/growth-arresting machinery. Much has been studied about the Tax gene of HTLV-1 in terms of tumorigenicity and targeted therapy, but these results prompted us to consider HBZ as a novel target for clinical intervention in the treatment of ATL.

T-cell-derived soluble factors that inhibit both X4 and R5 HIV are recognized as important in controlling HIV. Whereas three β chemokines, regulated-on-activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β, account for the suppression of R5 HIV by blockade of HIV entry, the major components responsible for the inhibition of X4 HIV strains have not been identified previously. We identify these factors primarily as a mixture of three β chemokines [macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), and I-309] and two RNases (angiogenin and RNase 4) of lesser potency and show that in a clade B population, some correlate with clinical status and are produced by both CD4+ and CD8+ T cells (chemokines, angiogenin) or only by CD8+ T cells (RNase 4). The antiviral mechanisms of these HIV X4-suppressive factors differ from those of the previously described HIV R5-suppressive β chemokines.

Control of persistent viral infection relies particularly on cell-mediated immunity comprised of CD4+ and CD8+ T cells. There is accumulating evidence on the relevance of soluble factor(s) secreted by CD8+ and CD4+ T cells in controlling HIV replication in vivo. While the β chemokines RANTES, MIP1-α and MIP1-β collectively account for the suppression of R5 viruses, yet information is lacking on the identity of the molecules involved in the suppression of X4 viruses. Proteins that inhibit the replication of X4 HIV isolates were purified from the conditioned media (CM) of immortalized CD8+ and CD4+ T cell lines from HIV+ long-term non-progressors subjects (LTNPs) and identified as the β chemokines macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) and I-309. These chemokines are secreted primarily by CD4+ T cells but also by CD8+T cells. CD4+ T cells of asymptomatic HIV+ individuals secreted significantly higher levels of MDC and TARC compared with subjects who progressed to AIDS. Recombinant human MDC, TARC and I309 induced a dose dependent inhibition of X4 viruses. A cocktail of neutralizing antibodies against MDC, TARC and I309 abrogated the inhibition of the replication of X4 viruses mediated by the endogenous chemokines in PBMC and CD8-depleted PBMC cells acutely infected in vitro. While the β chemokines RANTES, MIP1-α and MIP1-β suppress R5 viruses by blocking their entry into host cells the mechanism of inhibition of X4 viruses mediated by MDC, TARC and I-309 is a post entry mechanism of suppression. These molecules represent a major component of the soluble anti-X4 activity of T cells, suggesting that the mechanism whereby CD8+ and CD4+ T cells contribute to the control of HIV-1 replication may relate to the secretion of MDC, TARC and I-309.These results may be relevant to HIV pathogenesis.

Control of persistent viral infection relies particularly on cell-mediated immunity comprised of CD4+ and CD8+ T cells. There is accumulating evidence on the relevance of soluble factor(s) secreted by CD8+ and CD4+ T cells in controlling HIV replication in vivo. While the α chemokines RANTES, MIP1-α and MIP1-β collectively account for the suppression of R5 viruses, yet information is lacking on the identity of the molecules involved in the suppression of X4 viruses. Proteins that inhibit the replication of X4 HIV isolates were purified from the conditioned media (CM) of immortalized CD8+ and CD4+ T cell lines from HIV+ long-term non-progressors subjects (LTNPs) and identified as the α chemokines macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) and I309. These chemokines are secreted primarily by CD4+ T cells but also by CD8+T cells. CD4+ T cells of asymptomatic HIV+ individuals secreted significantly higher levels of MDC and TARC compared with subjects who progressed to AIDS. Recombinant human MDC, TARC and I309 induced a dose dependent inhibition of X4 viruses. A cocktail of neutralizing antibodies against MDC, TARC and I309 abrogated the inhibition of the replication of X4 viruses mediated by the endogenous chemokines in a dose dependent manner in PBMC and CD8-depleted PBMC cells acutely infected in vitro. These studies demonstrate that MDC, TARC and I309 represent a major component of the soluble anti-X4 activity of T cells, suggesting that the mechanism whereby CD8+ and CD4+ T cells contribute to the control of HIV-1 replication may relate to the secretion of these molecules.

Human neutrophil alpha-defensin 4 (HNP4) is more effective than HNP1-3 in protecting human peripheral blood mononuclear cells from infection by both X4 and R5 HIV-1 strains. HNP4 binds to both CD4 and gp120 approximately two orders of magnitude weaker than does HNP1, and is less effectively sequestered by glycosylated serum proteins than HNP1. These results suggest that the HIV-1 inhibition by HNP4 stems at least partially from a unique and lectin-independent property of HNP4 with CD4 and/or gp120. Our finding identifies an anti-HIV-1 property of HNP4 and may have implications in the development of new antiviral agents for AIDS therapy.

Entry of herpes simplex virus (HSV) 1 into cells requires the interaction of HSV gD with herpesvirus entry mediator or nectin1 receptors, and fusion with cell membrane mediated by the fusion glycoproteins gB, gH, and gL. We report that the gD ectodomain in soluble form (amino acids 1-305) was sufficient to rescue the infectivity of a gD-null HSV mutant, indicating that gD does not need to be anchored to the virion envelope to mediate entry. Entry mediated by soluble gD required, in addition to the receptor-binding sites contained within residues 1-250, a discrete downstream portion (amino acids 261-305), located proximal to the transmembrane segment in full-length gD. We named it as profusion domain. The pro-fusion domain was required for entry mediated by virion-bound gD, because its substitution with the corresponding region of CD8 failed to complement the infectivity of gD(-/+) HSV. Furthermore, a receptor-negative gD (gD(Delta6-259)) inhibited virus infectivity when coexpressed with wild-type gD; i.e., it acted as a dominant-negative gD mutant. The pro-fusion domain is proline-rich, which is characteristic of regions involved in protein-protein interactions. P291L-P292A substitutions diminished the gD capacity to complement gD(-/+) HSV infectivity. We propose that gD forms a tripartite complex with its receptor and, by way of the proline-rich pro-fusion domain, with the fusion glycoproteins, or with one of them. The tripartite complex would serve to recruit/activate the fusion glycoproteins and bring them from a fusion-inactive to a fusion-active state, such that they execute fusion of the virion envelope with cell membrane.

The herpes simplex virus type 1(JMP) [HSV-1(JMP)] mutant was selected for its ability to grow and form plaques in receptor-negative J cells. It enters J cells through a novel gD-dependent pathway, independent of all known HSV receptors, nectin1, nectin2, and HveA. Evidence that the pathway is dependent on a nectin3 binding site on HSV-1(JMP) and requires three mutations in gD rests on the following. We derived monoclonal antibodies to nectin3 and show that J cells express nectin3. HSV-1(JMP) entry and cell-to-cell spread were inhibited by soluble nectin3-Fc, demonstrating that virions carry a binding site for nectin3. The site is either directly involved in HSV-1(JMP) entry, or nectin3 binding to its site affects the gD domains involved in entry (entry site). HSV-1(JMP) entry and cell-to-cell spread in J cells were also inhibited by soluble nectin1-Fc, showing that the nectin1 binding site on gD(JMP) overlaps with the entry site or that nectin1 binding to gD affects the entry site. gD(JMP) carries three mutations, S140N, R340H, and Q344R. The latter two lie in the C tail and are present in the parental HSV-1(MP). HSV-1 strain R5000 carrying the S140N substitution was not infectious in J cells, indicating that this substitution was not sufficient. We constructed two recombinants, one carrying the three substitutions and the other carrying the two C-tail substitutions. Only the first recombinant infected J cells with an efficiency similar to that of HSV-1(JMP), indicating that the three mutations are required for the novel entry pathway. The results highlight plasticity in gD which accounts for changes in receptor usage.

CD4+ T cells are required for immunity against many viral infections, including HIV-1 where a positive correlation has been observed between strong recall responses and low HIV-1 viral loads. Some HIV-1-specific CD4+ T cells are preferentially infected with HIV-1, whereas others escape infection by unknown mechanisms. One possibility is that some CD4+ T cells are protected from infection by the secretion of soluble HIV-suppressive factors, although it is not known whether these factors are produced during primary antigen-specific responses. Here, we show that soluble suppressive factors are produced against CXCR4 and CCR5 isolates of HIV-1 during the primary immune response of human CD4+ T cells. This activity requires antigenic stimulation of naïve CD4+ T cells. One anti-CXCR4 factor is macrophage-derived chemokine (chemokine ligand 22, CCL22), and anti-CCR5 factors include macrophage inflammatory protein-1 alpha (CCL3), macrophage inflammatory protein-1 beta (CCL4), and RANTES (regulated upon activation of normal T cells expressed and secreted) (CCL5). Intracellular staining confirms that CD3+CD4+ T cells are the source of the prototype HIV-1-inhibiting chemokines CCL22 and CCL4. These results show that CD4+ T cells secrete an evolving HIV-1-suppressive activity during the primary immune response and that this activity is comprised primarily of CC chemokines. The data also suggest that production of such factors should be considered in the design of vaccines against HIV-1 and as a mechanism whereby the host can control infections with this virus.

The site on nectin1 receptor required for herpes simplex virus (HSV) entry into the cell was previously mapped to the 64-94 region, encompassing the predicted CC'C" region of the immunoglobulin V domain. Within it lies a minimal HSV entry site (residues 77-94). Here we transferred the 65-76 region (C strand and CC' loop) and portions, or single amino acids, thereof to nectin2, a homolog nonfunctional for wt HSV-1 entry. Replacement of the seven- or of three-amino-acid-long stretches from nectin1 to nectin2 (amino acids 69-75, 69-71, or 72-75) transferred wt HSV-1 and BHV-1 entry activity and enhanced HSV-2, PrV, and HSV-HSV(U21) entry to levels observed with nectin1. Thus, the CC' ridge is sufficient to mediate wt HSV entry at a reduced level and responsible for the wide virus range of the receptor. Altogether the HSV entry site appears to be composed of contiguous synergistic regions, 64-76 and 77-94, each independently capable of mediating virus entry at reduced efficiency.

ABSTRACT A novel member of the nectin family, nectin1γ, was molecularly cloned. The cDNA has the same ectodomain as nectin1α and nectin1β, the two known transmembrane isoforms that serve as receptors for herpes simplex virus (HSV) entry into human cell lines (nectin1α and nectin1β, also called PRR1-HveC and HIgR, respectively). The 1.4-kb transcript, which originated by alternative splicing, is expressed in human cell lines, and appears to have a narrow distribution in human tissues. The sequence does not have a hydrophobic anchoring region, and the protein is secreted in the culture medium of cells transfected with the cDNA. Nectin1γ, purified from culture medium, can compete with membrane-bound nectin1β and reduce HSV infectivity. The expression of nectin1γ cDNA in cells resistant to HSV infection and lacking HSV receptors enables HSV to enter the cell, which implies that it is present at the cell surface. Thus, nectin1γ has the potential both to mediate and to reduce HSV entry into cells.

Human nectin1 (hNectin1), an adhesion molecule belonging to the nectin family of the immunoglobulin superfamily, mediates entry of herpes simplex virus (HSV) into cells. The hNectin1 domain that mediates virus entry into cells and also binds glycoprotein D (gD) has been localized to the first N-terminal V-type domain. The poliovirus receptor (PVR) is a structural homolog to nectins, but it cannot function as an HSV entry receptor. hNectin1-PVR chimeras were constructed to functionally locate the site on hNectin1 involved in HSV entry (HSV entry site). The epitope recognized by monoclonal antibody (MAb) R1.302, which is able to block HSV entry, was also located. The chimeric receptors were designed to preserve the overall structure of the V domain. The HSV entry activity mapped entirely to the hNectin1 portion located between residues 64 and 94 (64-94), likely to encode the C, C', and C" beta -strands and intervening loops. In turn, this site consisted of two portions: one with low-level basal activity for HSV entry (77-94), and one immediately upstream (residues 64 to 76) which greatly enhanced the HSV entry activity of the downstream region. The gD-binding site mapped substantially to the same site, whereas the MAb R1.302 epitope also required a further downstream portion (95-102). The involvement of the 64-76 portion is at difference with previous indirect mapping results that were based on competitive binding studies (C. Krummenacher et al., J. Virol. 74:10863-10872, 2000). The A, A', B, D, E, F, and G beta -strands and intervening loops did not appear to play any role in HSV entry. According to the predicted three-dimensional structure of PVR, the C C' C" site is located peripherally in the V domain and very likely represents an accessible portion at the cell surface.