Bacterial DnaK reduces the activity of anti-cancer drugs cisplatin and 5FU.

BACKGROUND: Chemotherapy is a primary treatment for cancer, but its efficacy is often limited by cancer-associated bacteria (CAB) that impair tumor suppressor functions. Our previous research found that Mycoplasma fermentans DnaK, a chaperone protein, impairs p53 activities, which are essential for most anti-cancer chemotherapeutic responses. METHODS: To investigate the role of DnaK in chemotherapy, we treated cancer cell lines with M. fermentans DnaK and then with commonly used p53-dependent anti-cancer drugs (cisplatin and 5FU). We evaluated the cells' survival in the presence or absence of a DnaK-binding peptide (ARV-1502). We also validated our findings using primary tumor cells from a novel DnaK knock-in mouse model. To provide a broader context for the clinical significance of these findings, we investigated human primary cancer sequencing datasets from The Cancer Genome Atlas (TCGA). We identified F. nucleatum as a CAB carrying DnaK with an amino acid composition highly similar to M. fermentans DnaK. Therefore, we investigated the effect of F. nucleatum DnaK on the anti-cancer activity of cisplatin and 5FU. RESULTS: Our results show that both M. fermentans and F. nucleatum DnaKs reduce the effectiveness of cisplatin and 5FU. However, the use of ARV-1502 effectively restored the drugs' anti-cancer efficacy. CONCLUSIONS: Our findings offer a practical framework for designing and implementing novel personalized anti-cancer strategies by targeting specific bacterial DnaKs in patients with poor response to chemotherapy, underscoring the potential for microbiome-based personalized cancer therapies.

Mycoplasma DnaK expression increases cancer development in vivo upon DNA damage.

Well-controlled repair mechanisms are involved in the maintenance of genomic stability, and their failure can precipitate DNA abnormalities and elevate tumor risk. In addition, the tumor microenvironment, enriched with factors inducing oxidative stress and affecting cell cycle checkpoints, intensifies DNA damage when repair pathways falter. Recent research has unveiled associations between certain bacteria, including Mycoplasmas, and various cancers, and the causative mechanism(s) are under active investigation. We previously showed that Mycoplasma fermentans DnaK, an HSP70 family chaperone protein, hampers the activity of proteins like PARP1 and p53, crucial for genomic integrity. Moreover, our analysis of its interactome in human cancer cell lines revealed DnaK's engagement with several components of DNA-repair machinery. Finally, in vivo experiments performed in our laboratory using a DnaK knock-in mouse model generated by our group demonstrated that DnaK exposure led to increased DNA copy number variants, indicative of genomic instability. We present here evidence that expression of DnaK is linked to increased i) incidence of tumors in vivo upon exposure to urethane, a DNA damaging agent; ii) spontaneous DNA damage ex vivo; and iii) expression of proinflammatory cytokines ex vivo, variations in reactive oxygen species levels, and increased ß-galactosidase activity across tissues. Moreover, DnaK was associated with increased centromeric instability. Overall, these findings highlight the significance of Mycoplasma DnaK in the etiology of cancer and other genetic disorders providing a promising target for prevention, diagnostics, and therapeutics.

Strain-specific alterations in gut microbiome and host immune responses elicited by tolerogenic Bifidobacterium pseudolongum.

The beneficial effects attributed to Bifidobacterium are largely attributed to their immunomodulatory capabilities, which are likely to be species- and even strain-specific. However, their strain-specificity in direct and indirect immune modulation remain largely uncharacterized. We have shown that B. pseudolongum UMB-MBP-01, a murine isolate strain, is capable of suppressing inflammation and reducing fibrosis in vivo. To ascertain the mechanism driving this activity and to determine if it is specific to UMB-MBP-01, we compared it to a porcine tropic strain B. pseudolongum ATCC25526 using a combination of cell culture and in vivo experimentation and comparative genomics approaches. Despite many shared features, we demonstrate that these two strains possess distinct genetic repertoires in carbohydrate assimilation, differential activation signatures and cytokine responses signatures in innate immune cells, and differential effects on lymph node morphology with unique local and systemic leukocyte distribution. Importantly, the administration of each B. pseudolongum strain resulted in major divergence in the structure, composition, and function of gut microbiota. This was accompanied by markedly different changes in intestinal transcriptional activities, suggesting strain-specific modulation of the endogenous gut microbiota as a key to immune modulatory host responses. Our study demonstrated a single probiotic strain can influence local, regional, and systemic immunity through both innate and adaptive pathways in a strain-specific manner. It highlights the importance to investigate both the endogenous gut microbiome and the intestinal responses in response to probiotic supplementation, which underpins the mechanisms through which the probiotic strains drive the strain-specific effect to impact health outcomes.

Characterization of the interactome profiling of Mycoplasma fermentans DnaK in cancer cells reveals interference with key cellular pathways.

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.

Comparison of SARS-CoV-2 Receptors Expression in Primary Endothelial Cells and Retinoic Acid-Differentiated Human Neuronal Cells.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is primarily responsible for coronavirus disease (COVID-19) and it is characterized by respiratory illness with fever and dyspnea. Severe vascular problems and several other manifestations, including neurological ones, have also been frequently reported, particularly in the great majority of "long hauler" patients. SARS-CoV-2 infects and replicates in lung epithelial cells, while dysfunction of endothelial and neuronal brain cells has been observed in the absence of productive infection. It has been shown that the Spike protein can interact with specific cellular receptors, supporting both viral entry and cellular dysfunction. It is thus clear that understanding how and when these receptors are regulated, as well as how much they are expressed would help in unveiling the multifaceted aspects of this disease. Here, we show that SH-SY5Y neuroblastoma cells express three important cellular surface molecules that interact with the Spike protein, namely ACE2, TMPRSS2, and NRP1. Their levels increase when cells are treated with retinoic acid (RA), a commonly used agent known to promote differentiation. This increase matched the higher levels of receptors observed on HUVEC (primary human umbilical vein endothelial cells). We also show by confocal imaging that replication-defective pseudoviruses carrying the SARS-CoV-2 Spike protein can infect differentiated and undifferentiated SH-SY5Y, and HUVEC cells, although with different efficiencies. Neuronal cells and endothelial cells are potential targets for SARS-CoV-2 infection and the interaction of the Spike viral protein with these cells may cause their dysregulation. Characterizing RNA and protein expression tempo, mode, and levels of different SARS-CoV-2 receptors on both cell subpopulations may have clinical relevance for the diagnosis and treatment of COVID-19-infected subjects, including long hauler patients with neurological manifestations.

Combined cART including Tenofovir Disoproxil, Emtricitabine, and Dolutegravir has potent therapeutic effects in HIV-1 infected humanized mice.

HIV-1 reservoirs persist in the presence of combined antiretroviral therapy (cART). However, cART has transformed HIV-1 infection into a chronic disease marked by control of HIV-1 viral load and mortality reduction. Major challenges remain, including viral resistance upon termination of cART and persistence and identification of tissue distribution of HIV-1 reservoirs. Thus, appropriate animal models that best mimic HIV-1 pathogenesis are important, and the current study complements our previously published validation of the CD34+ hematopoietic humanized mouse model for this purpose. Here we analyze viral suppression using the recently developed combination of antiretrovirals that include Tenofovir Disoproxil (TDF), Emtricitabine (FTC), and Dolutegravir (DTG), a choice based on recent clinical outcomes showing its improved antiretroviral potency, CD4+ T cell preservation, tolerability, and prevention of viral drug resistance compared to that of previous regimens. We used quantitative Airyscan-based super resolution confocal microscopy of selected mouse tissues. Our data allowed us to identify specific solid tissue reservoirs of human T cells expressing the HIV-1 core protein p24. In particular, lymph node, brain, spleen, and liver were visualized as reservoirs for residual infected cells. Marked reduction of viral replication was evident. Considering that detection and visualization of cryptic sites of HIV-1 infection in tissues are clearly crucial steps towards HIV-1 eradication, appropriate animal models with pseudo-human immune systems are needed. In fact, current studies with humans and non-human primates have limited sample availability at multiple stages of infection and cannot easily analyze the effects of differently administered combined antiretroviral treatments on multiple tissues. That is easier to manage when working with humanized mouse models, although we realize the limitations due to low human cell recovery and thus the number of cells available for thorough and comprehensive analyses. Nonetheless, our data further confirm that the CD34+ humanized mouse model is a potentially useful pre-clinical model to study and improve current anti-HIV-1 therapies.

Role of Mycoplasma Chaperone DnaK in Cellular Transformation.

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.

Seroprevalence of HSV-1 and 2 in HIV-infected males with and without GUD: Study from a tertiary care setting of India.

BACKGROUND: Herpes simplex virus (HSV) infection is associated with an increased risk of both human immune deficiency virus (HIV) transmission and acquisition. However, in India, literature on HSV infections in in HIV-infected males has been scarce. The present study was carried out to assess the seroprevalence of these viruses in HIV-infected males, so as to provide a baseline data from India and report on HSV associated GUD prevalence in HIV infected males. OBJECTIVE: The aim of the study was to estimate the seroprevalence of herpes simplex type 1 and 2 viruses in HIV-infected males with and without genital ulcers disease (GUD). MATERIAL AND METHODS: It was a prospective study. We included a total of 351 male participants in this study. Among these 233 were HIV-infected and 118 HIV-uninfected males who served as controls. The seroprevalence was estimated, using HSV-1 and 2 type specific IgG and IgM antibodies by ELISA. RESULTS: HIV-infected patients had a median age of 32 ± 6.97 years (interquartile range: 28-36). Of the 351 males, 25.92% (91/351) were infected with HSV-1 and HSV-2 both. The overall seroprevalence of HSV-1 singly infected, HSV-2 singly infected, and dual infection in HIV-infected males was 39.92%, 25.58%, and 37.33% whereas in HIV-uninfected group the corresponding figures were 71.18%, 5.08%, and 3.38%, respectively. Seven of 233 (3%) HIV-infected males were having incident HSV infection. GUD was reported in both HSV-1 and HSV-2 seropositive individuals. CONCLUSIONS: Both HSV-1 and HSV-2 infections were found to be associated with GUD in HIV-infected patients. The prevalence of HIV-HSV co-infection among GUD patients is high.

Human Herpesviruses as Copathogens of HIV Infection, Their Role in HIV Transmission, and Disease Progression.

Of eight human herpesviruses (HHVs), often, only herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) find mention in medical literature as both of these viruses are commonly associated with genital lesions and oral ulcers, commonly known as cold sores. However, role of human herpesviruses as copathogens and in aggravation and in the transmission of other human diseases, especially the Acquired immunodeficiency syndrome (HIV/AIDS) has only very recently been recognized. Therefore, screening and treating subclinical HHV infections may offer slowing of HIV infection, disease progression, and its transmission. Beside HSV-1 and HSV-2, HHV-3 a causative agent of herpes zoster remained one of the first manifestations of HIV disease before the era of highly active antiretroviral therapy (HAART). HHV-5 also known as human Cytomegalovirus infection remains a significant risk factor for HIV-associated mortality and morbidity even in HAART era. It is proposed that Cytomegalovirus viremia could be a better predictor of HIV disease progression than CD4+ T-lymphocyte count. The role of HHV-4 or Epstein-Burr virus and HHV-6, HHV-7, and HHV-8 is still being investigated in HIV disease progression. This review provides insight into the current understanding about these 8 HHVs, their co-pathogenesis, and role in HIV/AIDS disease progression. The review also covers recent literature in favor and against administering anti-HHV treatment along with HAART for slower AIDS progression and interrupted sexual transmission.

Seroprevalence and determinants of Kaposi sarcoma-associated human herpesvirus 8 in Indian HIV-infected males.

In India Kaposi's sarcoma is rarely seen in AIDS patients. Hence the current belief is that the incidence of human herpesvirus-8 (HHV-8) is very low in this subcontinent, most probably due to the heterosexual route of HIV transmission. However, there is a scarcity of data on the prevalence of HHV-8 in India. In India the primary mode of HIV transmission is the heterosexual route. Therefore we aimed to determine the prevalence of antibodies against HHV-8 in North Indian HIV-infected men naive of antiretroviral therapy (ART). In a prospective study, 165 Indian adult males were recruited from an ART clinic. Blood samples were collected before administering any antiretroviral drug. The sera were tested for antibodies against HHV-8 using a commercial enzyme-linked immunosorbent assay (ELISA) kit, which detects IgG antibodies to lytic antigens of HHV-8. All positive samples were confirmed for the presence of anti-HHV-8 antibodies using an indirect immunofluorescence assay (IFA). The IFA kit is intended to detect primary, latent, persistent, or reactivated infection of HHV-8. Of the 165 males, 43 (26.06%) were positive by ELISA while 26 (15.8%) were also positive by IFA. Seroprevalence decreased with increasing age (p<0.05). Factors independently associated with HHV-8 infection were younger age group and alcohol consumption. These findings suggest that even in a heterosexual population, HHV-8 can be transmitted frequently.

Serologic prevalence of Toxoplasma gondii in Indian women of child bearing age and effects of social and environmental factors.

BACKGROUND: Seroprevalence and incidence of toxoplasmosis in women of child bearing age has remained a contentious issue in the Indian subcontinent. Different laboratories have used different patient recruitment criteria, methods and variable results, making these data difficult to compare. AIM: To map the point-prevalence and incidence of toxoplasmosis in India. MATERIAL AND METHODS: In this cross-sectional study, a total of 1464 women of fertile age were recruited from 4 regions using similar recruitment plans. This included women from northern (203), southern (512), eastern (250) and western (501) regions of India. All samples were transported to a central laboratory in Delhi and tested using VIDAS technology. Their age, parity, eating habits and other demographic and clinical details were noted. RESULTS: Most women were in the 18-25 years age group (48.3%), followed by 26-30 years (28.2%) and 31-35 years (13.66). Few (45) women older than 35 yr. were included. Overall prevalence of anti-Toxoplasma IgG antibodies was seen in 22.40%, with significantly more in married women (25.8%) as compared to single women (4.3%). Prevalence increased steadily with age: 18.1% in the 18-25 yr. age group to 40.5% in women older than 40 yr. The prevalence was high (66%) in those who resided in mud houses. Region-wise, the highest prevalence was observed in South India (37.3%) and the lowest (8.8%) in West Indian women. This difference was highly significant (P<0.001). Prevalence was 21.2% in East India and 19.7% in North India. The IgM positivity rate ranged from 0.4% to 2.9% in four study centers. CONCLUSIONS: This pan-India study shows a prevalence rate of 22.4% with a wide variation in four geographical regions ranging from as low as 8.8% to as high as 37.3%. The overall IgM positivity rate was 1.43%, indicating that an estimated 56,737-176,882 children per year are born in India with a possible risk of congenital toxoplasmosis.

HIV-1 imposes rigidity on blood and semen cytokine networks.

PROBLEM: Although it is established that the levels of individual cytokines are altered by HIV-1 infection, the changes in cytokine interrelations that organize them into networks have been poorly studied. Here, we evaluated these networks in HIV-infected and HIV-uninfected individuals in fluid compartments that are critical for HIV-1 pathogenesis and transmission, namely blood and semen. METHOD OF STUDY: In samples collected from therapy-naïve HIV-1-infected and HIV-1-uninfected individuals, we measured HIV-1-load, CD4 cell count, and levels of 21 cytokines using a multiplex bead assay. RESULTS: Cytokine networks in blood and semen were different for HIV-1-infected and HIV-1-uninfected individuals. In both compartments of HIV-1-infected individuals, the cytokine networks were more interlocked than in controls: HIV-1 infection resulted in the establishment of new correlations and in the strengthening of pre-existing correlations between different cytokines. In blood and semen of HIV-infected patients, there were, respectively, 68 and 72 statistically significant correlations between cytokines, while in uninfected individuals, there were 18 and 21 such correlations. CONCLUSION: HIV-1 infection reorganizes the cytokine networks, establishing new strong correlations between various cytokines and thus imposes a high rigidity on the cytokine network. This rigidity may reflect the impairment of the ability of the immune system to respond to microbial challenges.

AIDS associated tuberculosis: a catastrophic collision to evade the host immune system.

Human immunodeficiency virus (HIV) is a causative agent of AIDS while Mycobacterium tuberculosis causes human tuberculosis, independently. HIV and M. tuberculosis co-infection is an intriguing immunopathological phenomenon. The effect is not simply an additive but far more than that. This review gives an account of how various host and pathogen specific factors interplay to make this co-infection one of the worst co-infection, rightly named as medical "double jeopardy". We have attempted to recount some of the immune mechanisms how both these pathogens disturb the balance of host immune system resulting into defects in the host phagocytic response, leading to apoptosis and chemokine dysregulation. The HIV provides protected shelter to the M. tuberculosis and M. tuberculosis provides conducive atmosphere through the interplay of various chemokines. We also touch upon the treatment associated complications like Immune reconstitution inflammatory syndrome (IRIS) these patients face.

Exploiting the anti-HIV-1 activity of acyclovir: suppression of primary and drug-resistant HIV isolates and potentiation of the activity by ribavirin.

Multiple clinical trials have demonstrated that herpes simplex virus 2 (HSV-2) suppressive therapy using acyclovir (ACV) or valacyclovir in HIV-1/HSV-2-infected persons increased the patient's survival and decreased the HIV-1 load. It has been shown that the incorporation of ACV-monophosphate into the nascent DNA chain instead of dGMP results in the termination of viral DNA elongation and directly inhibits laboratory strains of HIV-1. We evaluated here the anti-HIV activity of ACV against primary HIV-1 isolates of different clades and coreceptor specificity and against viral isolates resistant to currently used drugs, including zidovudine, lamivudine, nevirapine, a combination of nucleoside reverse transcriptase inhibitors (NRTIs), a fusion inhibitor, and two protease inhibitors. We found that, at clinically relevant concentrations, ACV inhibits the replication of these isolates in human tissues infected ex vivo. Moreover, addition of ribavirin, an antiviral capable of depleting the pool of intracellular dGTP, potentiated the ACV-mediated HIV-1 suppression. These data warrant further clinical investigations of the benefits of using inexpensive and safe ACV alone or in combination with other drugs against HIV-1, especially to complement or delay highly active antiretroviral therapy (HAART) initiation in low-resource settings.

Semen of HIV-1-infected individuals: local shedding of herpesviruses and reprogrammed cytokine network.

BACKGROUND: Semen is the main carrier of sexually transmitted viruses, including human immunodeficiency virus type 1 (HIV-1). However, semen is not just a mere passive transporter of virions but also plays an active role in HIV-1 transmission through cytokines and other biological factors. METHODS: To study the relationship between viruses and the chemokine-cytokine network in the male genital tract, we measured the concentrations of 21 cytokines/chemokines and the loads of HIV-1 and of 6 herpesviruses in seminal and blood plasma from HIV-1-infected and HIV-uninfected men. RESULTS: We found that (1) semen is enriched in cytokines and chemokines that play key roles in HIV-1 infection or transmission; (2) HIV-1 infection changes the chemokine-cytokine network in semen, further enriching it in cytokines that modulate its replication; (3) HIV-1 infection is associated with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) compartmentalized seminal reactivation; (4) CMV and EBV concomitant seminal shedding is associated with higher HIV-1 loads in blood and seminal plasma; and (5) CMV seminal reactivation increases the seminal levels of the CCR5 ligands RANTES and eotaxin, and of the CXCR3 ligand monokine induced by gamma interferon (MIG). CONCLUSIONS: HIV-1 infection results in an aberrant production of cytokines and reactivation of EBV and CMV that further changes the seminal cytokine network. The altered seminal milieu in HIV-1 infection may be a determinant of HIV-1 sexual transmission.