Infected Mice Research Articles

Identification of Brucella RS15060 as a novel type IV secretion system effector associated with bacterial virulence

Brucella is an intracellular parasitic pathogen that causes the worldwide zoonotic disease brucellosis. The type IV secretion system (T4SS) is utilized to secrete various effectors to help Brucella form Brucella-containing vacuoles within the cell and accomplish intracellular trafficking and replication. Brucella has fewer recognized effector proteins than other intracellular parasites in the Proteobacteria, indicating that Brucella may contain a large number of unidentified effector proteins. In this study, the optimal conditions for inducing protein secretion from Brucella were screened, and the secreted proteins of 2308 and the T4SS-deficient mutant SV123 under optimal conditions were collected for comparative proteomics analysis. By label-free quantitative proteomics, we identified 15 differential proteins. Through the β-lactamase TEM1 assay and indirect immunofluorescence assay, we identified RS15060 and RS10635 as novel T4SS effectors. Furthermore, by constructing mutation strains and performing cell/mouse infection experiments, we found that deletion of the rs15060 gene reduced the capacity of Brucella to replicate in cells and cause chronic infection in mice. In conclusion, a novel Brucella T4SS effector protein, RS15060, was identified to be associated with virulence in this study, and the discovery of effector proteins is conducive to a more comprehensive elucidation of T4SS function as well as to uncovering the cryptic strategies of Brucella survival in cells.

The predominant role of FliC contributes to the flagella-related pathogenicity of ST34 S. Typhimurium monophasic variant

Over the past two decades, the monophasic variant of Salmonella enterica serovar Typhimurium (S. Typhimurium) has rapidly emerged and increased worldwide. This upsurge is especially true for the European clone of the ST34 S. Typhimurium monophasic variant. The key distinction between ST34 S. Typhimurium and its monophasic variant is that the genes that encode for second-phase flagellin (FljB) and the regions around it have been replaced with various multidrug resistance cassettes. To determine if the loss of fljB or the retention of fliC,-the gene coding for first-phase flagellin (FliC)-, would impact its pathogenicity, we constructed various mutations, including deletions of fljB, fliC, fliC/fljB, and strains where fliC was replaced with fljB. Our results showed that the loss of fljB in ST34 S. Typhimurium and its monophasic variant does not affect bacterial motility, cell infection ability, survival in macrophages, induced pro-inflammatory cytokines secretion, virulence, or persistent infection in mice. However, the deletion of fliC caused a significant decrease in these outcomes for both strains, while the replacement of fliC with fljB only partially restored these capabilities. Consequently, we determined that FliC is predominant in the flagellar expression of ST34 S. Typhimurium other than FljB. This finding demonstrates that replacing the fljB gene with various resistance regions in ST34 S. Typhimurium monophasic variants can enhance bacterial survival under specific antibiotic farming practices and spread globally.

Metabolomic analysis reveals an important role of sphingosine 1-phosphate in the development of HFMD due to EV-A71 infection.

Hand, foot, and mouth disease (HFMD) is a serious pediatric infectious disease that causes immeasurable physical and mental health burdens. Currently, there is a lack of information on the mechanisms of HFMD severity and early diagnosis. We performed metabolomic profiling of sera from 84 Enterovirus A71 (EV-A71) infections and 45 control individuals. Targeted metabolomics assays were employed to further validate some of the differential metabolic molecules. We identified significant molecular changes in the sera of HFMD patients compared to healthy controls (HCs). A total of 54, 60, 35, and 62 differential metabolites were screened between mild cases and HCs, severe cases and HCs, severe cases and mild cases, and among the three groups, respectively. These differential metabolites implicated dysregulation of the tricarboxylic acid cycle, alanine, aspartate, and glutamate metabolism, and valine, leucine, and isoleucine biosynthesis. The diagnostic panel based on some overlapped differential metabolites could effectively discriminate severe cases from mild cases with an AUC of 0.912 (95% CI: 0.85-0.97) using the logistic regression model. Next, we found the elevation of serum sphingosine 1-phosphate (S1P) level in EV-A71 infection mice, which was similar to clinical observation. Importantly, after blocking the release of S1P by MK571, the clinical symptoms and survival of mice were significantly improved, involving the reduction of leukocyte infiltration in infected brain tissues. Collectively, our data provided a landscape view of metabolic alterations in EV-A71 infected children and revealed regulating S1P metabolism was an exploitable therapeutic target against EV-A71 infection.

A newly developed oral infection mouse model of shigellosis for immunogenicity and protective efficacy studies of a candidate vaccine.

Shigella infection poses a significant public health challenge in the developing world. However, lack of a widely available mouse model that replicates human shigellosis creates a major bottleneck to better understanding of disease pathogenesis and development of newer drugs and vaccines. BALB/c mice pre-treated with streptomycin and iron (FeCl3) plus desferrioxamine intraperitoneally followed by oral infection with virulent Shigella flexneri 2a resulted in diarrhea, loss of body weight, bacterial colonization and progressive colitis characterized by disruption of epithelial lining, loss of crypt architecture with goblet cell depletion, increased polymorphonuclear infiltration into the mucosa, submucosal swelling (edema), and raised proinflammatory cytokines and chemokines in the large intestine. To evaluate the usefulness of the model for vaccine efficacy studies, mice were immunized intranasally with a recombinant protein vaccine containing Shigella invasion protein invasion plasmid antigen B (IpaB). Vaccinated mice conferred protection against Shigella, indicating that the model is suitable for testing of vaccine candidates. To protect both Shigella and Salmonella, a chimeric recombinant vaccine (rIpaB-T2544) was developed by fusing IpaB with Salmonella outer membrane protein T2544. Vaccinated mice developed antigen-specific serum IgG and IgA antibodies and a balanced Th1/Th2 response and were protected against oral challenge with Shigella (S. flexneri 2a, Shigella dysenteriae, and Shigella sonnei) using our present mouse model and Salmonella (Salmonella Typhi and Paratyphi) using an iron overload mouse model. We describe here the development of an oral Shigella infection model in wild-type mouse. This model was successfully used to demonstrate the immunogenicity and protective efficacy of a candidate protein subunit vaccine against Shigella.

Vaccination with a Replication-Dead Murine Gammaherpesvirus Lacking Viral Pathogenesis Genes Inhibits WT Virus Infection

Gammaherpesviruses are oncogenic pathogens that establish lifelong infections. There are no FDA-approved vaccines against Epstein–Barr virus or Kaposi sarcoma herpesvirus. Murine gammaherpesvirus-68 (MHV68) infection of mice provides a system for investigating gammaherpesvirus pathogenesis and testing vaccine strategies. Prime-boost vaccination with a replication-dead virus (RDV) that does not express the essential replication and transactivator protein (RTA) encoded by ORF50 (RDV-50.stop) protected against WT virus replication and reduced latency in C57BL/6 mice, and prevented lethal disease in Ifnar1−/− mice. To further improve the RDV vaccine and more closely model KSHV vaccine design, we generated an RDV lacking the unique M1-M4 genes and the non-coding tRNA-miRNA-encoded RNAs (TMERs) 6, 7, and 8 that collectively promote latency of MHV68 in vivo. Prime-boost vaccination of mice with RDV-50.stop∆M1-M4 elicited neutralizing antibodies and virus-specific CD8 T-cell responses in the lungs and spleens, the respective sites of acute replication and latency, that were comparable to RDV-50.stop vaccination. When challenged with WT MHV68, vaccinated mice exhibited a near-complete block of lytic replication and a reduction in latency and reactivation. We conclude that the unique M1-M4 genes and TMERs 6, 7, and 8, which are major determinants of WT MHV68 pathogenesis, are not required for eliciting protective immunity.

Myeloid-derived suppressor cells inhibit responses of T follicular helper cells during experimental Plasmodium yoelii infection.

Malaria remains a significant global public health problem. T follicular helper (Tfh) cells, a subset of CD4+ T cells, have the capacity to regulate B cells, plasma cells, and antibody production, among other functions. Myeloid-derived suppressor cells (MDSCs) possess strong immunosuppressive abilities and can negatively regulate various immune responses. However, the role of MDSCs in inhibiting Tfh-cell responses during Plasmodium infection remains unclear. In this study, we investigated the regulatory effect of MDSCs on Tfh cell-mediated immune responses upon Plasmodium infection. We found that the numbers of MDSCs increased upon Plasmodium infection. Further mechanism study revealed that MDSC-derived Arg-1 and PD-L1 prevented Tfh cell proliferation and activation. Conversely, the addition of nor-NOHA or anti-PD-L1 monoclonal antibodies enhanced the proliferation and activation of Tfh cells, indicating that the inhibitory effect of MDSCs on Tfh cells was dependent on Arg-1 and PD-1/PD-L1. Invivo depletion of MDSCs enhanced Tfh-cell responses and antibody production, as well as relieved symptoms of infected mice and improved their survival rates. These findings provide insights into the immunosuppressive role of MDSCs in inhibiting Tfh cell immune responses and further impairing humoral immunity. Our study provides new strategies for malaria prevention and control.

Three transporters, including the novel Gai1 permease, drive amino acid uptake in Histoplasma yeasts

ABSTRACT The dimorphic fungus Histoplasma capsulatum, which almost exclusively resides within host phagocytic cells during infection, must meet its nutritional needs by scavenging molecules from the phagosome environment. The requirement for gluconeogenesis, but not fatty acid catabolism, for intracellular growth, implicates amino acids as a likely intracellular nutrient source. Consequently, we investigated Histoplasma growth on amino acids. Growth assays demonstrated that Histoplasma yeasts readily utilize most amino acids as nitrogen sources but only efficiently catabolize glutamine, glutamate, aspartate, proline, isoleucine, and alanine as carbon sources. An amino acid permease-based conserved domain search identified 28 putative amino acid transporters within the Histoplasma genome. We characterized the substrate specificities of the major Histoplasma amino acid transporters using a Saccharomyces cerevisiae heterologous expression system and found that H. capsulatum Dip5, Gap3, and a newly described permease, Gai1, comprise most of Histoplasma’s amino acid import capacity. Histoplasma yeasts deficient in these three transporters are impaired for growth on free amino acids but proliferate within macrophages and remain fully virulent during infection of mice, indicating that free amino acids are not the principal nutrient source within the phagosome to support Histoplasma proliferation during infection.

Comparative study of two Rift Valley fever virus field strains originating from Mauritania.

Rift Valley fever (RVF) is one of the major viral arthropod-borne diseases in Africa. In recent decades, RVF virus (RVFV), the causative agent of RVF, has been responsible for multiple outbreaks in West Africa with important consequences on human and animal health. In particular, an outbreak occurred in 2010 after heavy rains in the desertic region of Adrar, Mauritania. It was characterized by the appearance of severe clinical signs among dromedary camels. Another one occurred in 2013-2014 across Senegal and the southern part of Mauritania. In this study, we characterized two RVFV field strains isolated during these two outbreaks. The first strain, MRU25010-30, was isolated from a camel (2010) while the second, MRU2687-3, was isolated from a goat (2013). By deep-sequencing and rapid amplification of cDNA-ends by polymerase chain reaction, we successfully sequenced the complete genome of these two RVFV strains as well as the reference laboratory strain ZH548. Phylogenetic analysis showed that the two field viruses belong to two different RVFV genetic lineages. Moreover, we showed that MRU25010-30 replicates more efficiently in various in vitro cell culture models than MRU2687-3 and ZH548. In vivo, MRU25010-30 caused rapid death of BALB/c mice and proved to be more virulent than MRU2687-3, regardless of the route of inoculation (subcutaneous or intranasal). The virulence of MRU25010-30 is associated with a high viral load in the liver and serum of infected mice, while the death of mice infected with MRU2687-3 and ZH548 correlated with a high viral load in the brain. Altogether, the data presented in this study provide new avenues to unveil the molecular viral determinants that modulate RVFV virulence and replication capacity.

Modulation of 8-Oxoguanine DNA Glycosylase 1 (OGG1) Alleviated Anemia Severity and Excessive Cytokines Release during Plasmodium berghei Malaria in Mice

Background: The interplay of OGG1, 8-Oxoguanine, and oxidative stress triggers the exaggerated release of cytokines during malaria, which worsens the outcome of the disease. We aimed to investigate the involvement of OGG1 in malaria and assess the effect of modulating its activity on the cytokine environment and anemia during P. berghei malaria in mice. Methods: Plasmodium berghei ANKA infection in ICR mice was used as a malaria model. OGG1 concentration and oxidative stress levels in P. berghei-infected mice and their control counterparts were assessed during malaria using enzyme-linked immunosorbent assay. OGG1 activity in malaria mice was modulated using treatment with TH5487 and O8-OGG1 inhibitors. The effects of modulating OGG1 activity using OGG1 inhibitors on cytokine release and anemia during P. berghei malaria infection were assessed by cytometric bead array and measurement of total normal red blood cell count respectively. Results: The plasma OGG1 level was significantly upregulated and positively correlated with parasitemia during P. berghei malaria in mice. Modulation of OGG1 ameliorated malaria severity by improving the total normal RBC count in TH5487 and O8-treated mice. Modulation of OGG1 with TH5487 caused significant reductions in serum levels of TNF-α, IFN-γ, IL-6, and IL-10. Similarly, OGG1 modulation activity using an O8-OGG1 inhibitor caused a significant reduction in serum levels of TNF-α, IL-2, IL-6, and IL-10. Conclusion: The findings indicate the involvement of OGG1 in the P. berghei malaria infection. OGG1 inhibition by TH5487 and O8-OGG1 inhibitors suppressed excessive cytokine release, and this may represent a novel therapeutic strategy for ameliorating the severity of malaria infection.

Mn and Zn-Doped Multivariate Metal-Organic Framework as a Metalloimmunological Adjuvant to Promote Protection Against Tuberculosis Infection.

A first-in-class vaccine adjuvant delivery system, Mn-ZIF, is developed by incorporating manganese (Mn) into the zinc-containing zeolitic-imidazolate framework-8 (ZIF-8). The mixed metal approach, which allowed for tunable Mn doping, is made possible by including a mild reducing agent in the reaction mixture. This approach allows up to 50% Mn, with the remaining 50% Zn within the ZIF. This multivariate approach exhibits significantly decreased cytotoxicity compared to ZIF-8. The porous structure of Mn-ZIF enables the co-delivery of the STING agonist cyclic di-adenosine monophosphate (CDA) through post-synthetic loading, forming CDA@Mn-ZIF. The composite demonstrated enhanced cellular uptake and synergistic activation of the cGAS-STING pathway, producing proinflammatory cytokines and activating antigen-presenting cells (APCs). In a preclinical Mycobacterium tuberculosis (Mtb) model, CDA@Mn-ZIF formulates with the CysVac2 fusion protein elicited a potent antigen-specific T-cell response and significantly reduced the mycobacterial burden in the lungs of infected mice. These findings highlight the potential of CDA@Mn-ZIF as a promising adjuvant for subunit vaccines, offering a novel approach to enhancing vaccine efficacy and protection against infectious diseases such as tuberculosis.

Unique Leishmania mexicana clones secrete populations of extracellular vesicles with unique protein profile and variable infectious capability

The study of extracellular vesicles has become an incredibly important field of study, but the inherent heterogeneity of these vesicles continues to make their study challenging. The genetic variability and well-documented protocols for the growth and vesicle isolation from Leishmania parasites provide a unique opportunity to compare the heterogeneity of different populations secreted by Leishmania clones. Leishmania mexicana was cultured on solid SDM agar plates and 8 clonal colonies were selected. The EVs collected from the liquid cultures of these 8 clones were assessed by NTA, TEM, and proteomic analysis. We found that all 8 clonal L. mexicana cultures were visually indistinguishable from each other and had similar growth rate, and these physical similarities extended to their EVs. However, proteomic analysis reveals that the EVs collected have unique protein profiles compared to each other and EVs isolated from a heterogeneous liquid culture of L. mexicana. We selected 3 clonal EVs for further mouse infection experiments and found that EVs from CL7 L. mexicana consistently caused reduced footpad swelling in C57BL6 mice footpads compared to EVs from CL1, CL8, and heterogenous L. mexicana. This trend was not observed when infecting Balb/C mice and C57BL6 with the parasites alone, with only CL1 L. mexicana causing significantly increased infection in Balb/c mice. Our results together show that EVs isolated from different clonal colonies of L. mexicana have distinct differences in protein cargo which can lead to varying outcomes on Leishmania infection. Further evaluation will be needed to determine the underlying mechanisms behind this and verify that differences observed in infectivity are directly caused by variations between our L. mexicana clones, especially genetic sequencing and immunoblotting to validate our results.

THE EFFICACY OF THE AQUEOUS EXTRACT OF ADANSONIA DIGITATA AGAINST PLASMODIUM BERGHEI INFECTION IN MICE

The prevalence of malaria remains a significant threat in Sub-Saharan Africa. The occurrence of drug-resistant strains of Plasmodium in Nigeria underscores the urgent need for novel antimalarial medications. This research aims to assess the effectiveness of Adansonia digitata fruit pod extract against Plasmodium berghei infection in mice; determining the oral median lethal dose (LD50) and identifying phytochemical components using Lorke's and Evan's methodologies. The extract was assessed for its antiplasmodial activities against P. berghei-berghei in two murine malaria replicas using both suppressive and curative assays. Statistical analysis was conducted using one-way ANOVA. The LD50 of the extract through oral administration exceeded 5000 mg/kg. The extract contains alkaloids, saponins, flavonoids, phenols, tannins, and anthraquinones. The aqueous fruit pulp extract of A. digitata, when administered at 250, 500, and 1000 mg/kg of doses, exhibited a dose-dependent suppression of parasitemia by 25.70%, 42.57%, and 49.39% respectively compared to the control group, parasitaemia clearance rates was 31.76%, 43.34%, and 56.65% respectively. The parasitaemia clearance with a dosage of 1000 mg/kg was found to be statistically significant (p < 0.05).The reference drug (Chloroquine) administering a dosage of 5 mg/kg yielded a significant result (p < 0.05) parasitaemia clearance of 74.76%. Analysis of the Packed Cell Volume (PCV) levels revealed that those treated with the aqueous fruit pulp extracts of A. digitata at doses of 250, 500, and 1000 mg/kg maintained normal PCV values throughout suppressive and curative experiments. However, the PCV in the control groups was markedly diminished (p < 0.05) after both trials.

Comparison of a SARS-CoV-2 mRNA booster immunization containing additional antigens to a spike-based mRNA vaccine against Omicron BA.5 infection in hACE2 mice.

The emergence of SARS-CoV-2 variants presents challenges to vaccine effectiveness, underlining the necessity for next-generation vaccines with multiple antigens beyond the spike protein. Here, we investigated a multiantigenic booster containing spike and a chimeric construct composed of nucleoprotein (N) and membrane (M) proteins, comparing its efficacy to a spike-only booster against Omicron BA.5 in K18-hACE2 mice. Initially, mice were primed and boosted with Beta (B.1.351) spike-only mRNA, showing strong spike-specific T cell responses and neutralizing antibodies, albeit with limited cross-neutralization to Omicron variants. Subsequently, a spike-NM multiantigenic vaccine was then examined as a second booster dose for protection in hACE2-transgenic mice. Mice receiving either homologous spike-only or heterologous spike-NM booster had nearly complete inhibition of infectious virus shedding in oral swabs and reduced viral burdens in both lung and nasal tissues following BA.5 challenge. Examination of lung pathology further revealed that both spike-only and spike-NM boosters provided comparable protection against inflammatory infiltrates and fibrosis. Moreover, the spike-NM booster demonstrated neutralization efficacy in a pseudovirus assay against Wuhan-Hu-1, Beta, and Omicron variants akin to the spike-only booster. These findings indicate that supplementing spike with additional SARS-CoV-2 targets in a booster immunization confers equivalent immunity and protection against Omicron BA.5. This work highlights a promising strategy for individuals previously vaccinated with spike-only vaccines, potentially offering enhanced protection against emerging coronaviruses.

The TLR7/8 agonist INI-4001 enhances the immunogenicity of a Powassan virus-like-particle vaccine.

Powassan virus (POWV) is a pathogenic tick-borne flavivirus that causes fatal neuroinvasive disease in humans. There are currently no approved therapies or vaccines for POWV infection. Here, we develop a POW virus-like-particle (POW-VLP) based vaccine adjuvanted with the novel synthetic Toll-like receptor 7/8 agonist INI-4001. We demonstrate that INI-4001 outperforms both alum and the Toll-like receptor 4 agonist INI-2002 in enhancing the immunogenicity of a dose-sparing POW-VLP vaccine in mice. INI-4001 increases the magnitude and breadth of the antibody response as measured by whole-virus ELISA, induces neutralizing antibodies measured by FRNT, reduces viral burden in the brain of infected mice measured by RT qPCR, and confers 100% protection from lethal challenge with both lineages of POWV. We show that the antibody response induced by INI-4001 is more durable than standard alum, and 80% of mice remain protected from lethal challenge 9-months post-vaccination. Lastly, we show that the protection elicited by INI-4001 adjuvanted POW-VLP vaccine is unaffected by either CD4+ or CD8+ T cell depletion and can be passively transferred to unvaccinated mice indicating that protection is mediated through humoral immunity. This study highlights the utility of novel synthetic adjuvants in VLP-based vaccines.

Investigation of choline-binding protein of CbpD in the pathogenesis of Streptococcus suis type 2.

Streptococcus suis serotype 2 (S. suis type 2, SS2) is one of the zoonotic pathogens known to induce meningitis, septicemia, and arthritis in both pigs and humans, resulting in public health concerns. CbpD, also termed CrfP, is one of the choline-binding proteins (CBPs) that was found as a murein hydrolase in SS2 and plays crucial roles in natural genetic transformation under the control of ComRS-ComX regulatory system by a previous study. Nonetheless, the possible functions of CbpD in virulence and pathogenesis in SS2 remain unclear. In this study, a cbpD gene mutant (ΔcbpD) with its complemental strain (cΔcbpD) was constructed and further used to examine the pathogenic roles of CbpD in SS2 infection. The results showed that the CbpD deficiency leads to increased bacterial chain elongation and aggregation with little impact on the growth capability of SS2. The ΔcbpD strain represented more vulnerable to a thermo, acid, or oxidative stress. Elevated adhesion to human epithelial HEp-2 cells, decreased invasion into bEND3.0 cells, and more easily phagocytosed by murine RAW264.7 macrophages of ΔcbpD were found. The virulence of cbpD mutant was attenuated in a mouse infection model. Enhanced susceptibility within mice blood and impaired ability to colonize organs with alleviated histopathological lesions were also demonstrated as compared with wild-type SS2. It is noteworthy that the discrepant expression of multiple virulence-associated factors including serine/threonine phosphorylase Stp, anti-phagocytosis factor of transglutaminase TGase and adhesin of chaperon DnaJ, were examined resulting from the deletion of cbpD. Overall, these findings provided evidence that the CbpD factor contributes to SS2 infection and is involved in bacterial adhesion, invasion, and anti-phagocytosis processes by modulating crucial virulence-associated factors expression.

Necrosis drives susceptibility to Mycobacterium tuberculosis in Polg D257A mutator mice.

The genetic and molecular determinants that underlie the heterogeneity of Mycobacterium tuberculosis (Mtb) infection outcomes in humans are poorly understood. Multiple lines of evidence demonstrate that mitochondrial dysfunction can exacerbate mycobacterial disease severity and mutations in some mitochondrial genes confer susceptibility to mycobacterial infection in humans. Here, we report that mutations in mitochondria DNA (mtDNA) polymerase gamma (POLG) potentiate susceptibility to Mtb infection in mice. Polg D257A mutator mtDNA mice fail to mount a protective innate immune response at an early infection timepoint, evidenced by high bacterial burdens, reduced M1 macrophages, and excessive neutrophil infiltration in the lungs. Immunohistochemistry reveals signs of enhanced necrosis in the lungs of Mtb-infected Polg D257A mice and Polg D257A mutator macrophages are hyper-susceptible to extrinsic triggers of necroptosis ex vivo . By assigning a role for mtDNA mutations in driving necrosis during Mtb infection, this work further highlights the requirement for mitochondrial homeostasis in mounting balanced immune responses to Mtb.

4-Substituted-2-Thiazole Amides as Viral Replication Inhibitors of Alphaviruses.

2-(Methylthio)-N-(4-(naphthalen-2-yl)thiazol-2-yl)nicotinamide 1 was identified as an inhibitor against Chikungunya virus (CHIKV) with good antiviral activity [EC50 = 0.6 μM; EC90 = 0.93 μM and viral titer reduction (VTR) of 6.9 logs at 10 μM concentration] with no observed cytotoxicity (CC50 = 132 μM) in normal human dermal fibroblast (NHDF) cells. Structure-activity relationship (SAR) studies to further improve the potency, efficacy, and drug-like properties of 1 led to the discovery of a new potent inhibitor N-(4-(3-((4-cyanophenyl)amino)phenyl)thiazol-2-yl)-2-(methylthio)nicotinamide 26, which showed a VTR of 8.7 logs at 10 μM against CHIKV and an EC90 of 0.45 μM with considerably improved MLM stability (t1/2 = 74 min) as compared to 1. Mechanism of action studies show that 26 inhibits alphavirus replication by blocking subgenomic viral RNA translation and structural protein synthesis. The in vivo efficacy studies of compound 26 on CHIKV infection in mice are reported.

Identification and Analysis of Autophagy-Related Genes as Diagnostic Markers and Potential Therapeutic Targets for Tuberculosis Through Bioinformatics.

According to the World Health Organization, Mycobacterium tuberculosis infections affect approximately 25% of the world's population. There is mounting evidence linking autophagy and immunological dysregulation to tuberculosis (TB). As a result, this research set out to discover TB-related autophagy-related biomarkers and prospective treatment targets. We used five autophagy databases to get genes linked to autophagy and Gene Expression Omnibus databases to get genes connected to TB. Then, functional modules associated with autophagy were obtained by analyzing them using weighted gene co-expression network analysis. Both Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to examine the autophagy-related genes (ATGs) of important modules. Limma was used to identify differentially expressed ATGs (DE-ATGs), and the external datasets were used to further confirm their identification. We used DE-ATGs and a protein-protein interaction network to search the hub genes. CIBERSORT was used to estimate the kinds and amounts of immune cells. After that, we built a drug-gene interaction network and a network that included messenger RNA, small RNA, and DNA. At last, the differential expression of hub ATGs was confirmed by RT-qPCR, immunohistochemistry, and western blotting. The diagnostic usefulness of hub ATGs was evaluated using receiver operating characteristic curve analysis. Including 508 ATGs, four of the nine modules strongly linked with TB were deemed essential. Interleukin 1B (IL1B), CAPS1, and signal transducer and activator of transcription 1 (STAT1) were identified by intersection out of 22 DE-ATGs discovered by differential expression analysis. Research into immune cell infiltration found that patients with TB had an increased proportion of plasma cells, CD8 T cells, and M0 macrophages. A competitive endogenous RNA network utilized 10 long non-coding RNAs and 2 miRNAs. Then, the IL1B-targeted drug Cankinumad was assessed using this network. During bioinformatics analysis, three hub genes were validated in mouse and macrophage infection models. We found that IL1B, CASP1, and STAT1 are important biomarkers for TB. As a result, these crucial hub genes may hold promise as TB treatment targets.

Protective role of TRPV1+ nociceptive neurons communication to macrophages against T. cruzi infection in mice

Chagas’ disease is a life-threatening condition caused by Trypanosoma cruzi. Patients with chronic disease may develop gastrointestinal, neurological, or associated neuro-digestive dysfunctions. CNS invasion by T. cruzi can occur in the acute phase, and its presence in the brain and cerebrospinal fluid was reported. T. cruzi induces nociceptor neuron activation and pain. Nociceptive neurons and macrophage interact in diseases, and this neuroimmune communication has a pivotal role in disease outcome. We investigated, the role of TRPV1+ neurons in experimental T. cruzi infection in mice. T. cruzi forms were observed in the DRG and spinal cord in early stages of acute infection, and intrathecal administration of T. cruzi antigens into spinal cord induced pain. Trpv1 mRNA expression was increased in the DRG of infected mice and targeting TRPV1 reduced T. cruzi-induced pain. TRPV1 nociceptor ablation increased blood parasitemia while TRPV1 knockout mice presented 50% mortality upon infection in a normally non-lethal model. TRPV1 knockout also worsened clinical outcomes (hepatomegaly and megacecum), increased plasmatic Th2 cytokines and nitrite in cardiac tissue, and reduced heart leukocyte infiltration. Conditioned media of capsaicin-stimulated DRG neurons decreased macrophage internalization of T. cruzi, and CGRP receptor antagonism in infected mice reduced pain, increased early parasitemia and promoted 18% mortality. This indicates that soluble mediators released upon nociceptor activation such as CGRP increase macrophage ability to control disease outcome. These data unveil TRPV1+ neurons release CGRP to limit macrophage internalization of T. cruzi, triggering protective mechanisms against T. cruzi infection.

Protection induced by Streptococcus pneumoniae extracellular vesicles against nasal colonization and invasive infection in mice and the role of PspA

Diseases caused by Streptococcus pneumoniae (pneumococcus) produce a great impact on public health, killing about one million people annually despite available vaccines. Recent research has revealed that the pneumococcus produces extracellular vesicles (pEVs), which display selective cargo and hold potential for vaccine development. Here, we evaluated the immunogenicity and protective potential of pEVs derived from a non-encapsulated pneumococcal strain (R6) using murine models of pneumococcal colonization and invasive pneumonia. Characterization of the immune response revealed that while pEVs contain multiple virulence determinants, immunization with these nanoparticles only induces antibodies against a subset of them. Specifically, subcutaneous immunization elicits a high antibody response against PspA, a modest response against PrsA, and limited response against Ply, MalX and PsaA. The antibody response was further supported by agglutination studies, showing that sera from pEV immunized mice agglutinate pneumococci and that PspA contributes to this response in a strain-dependent manner. Subcutaneous immunization with pEVs provides protection in the invasive pneumonia model whereas nasal immunization results in one log reduction in pneumococcal colonization of the upper respiratory tract. Finally, PspA is a strong contributor to protection in the invasive model and provides a degree of protection even across heterologous families of PspA. We conclude that pEVs demonstrate potential for vaccine development, protecting across capsular types and providing some degree of protection across heterologous PspA variants.