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Tumor-associated macrophages (TAMs), which display a tumor-supportive M2 phenotype, are closely related to tumor growth and metastasis. The reprogramming of TAMs toward a tumoricidal M1 profile has emerged as an attractive strategy for cancer immunotherapy. In this study, we found that the intratumoral injection of PcrV protein, a component of the Pseudomonas aeruginosa type 3 secretion system, suppressed tumor growth and increased apoptosis, inducible nitric oxide synthase (iNOS) expression, and the percentage of M1-polarized TAMs in tumor tissues. Furthermore, the intratumoral injection of PcrV-primed macrophages exerted a similar tumoricidal effect. In vitro analyses revealed that PcrV reeducated TAMs toward an antitumoral M1 phenotype and augmented their nitric oxide (NO)-mediated cytotoxicity against cancer cells. Mechanistically, we found that these effects were dependent on the activation of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)-mediated regulation of a PI3K/AKT/mTOR-glycolysis-NO feedback loop via direct interaction with TLR4. Collectively, these results revealed a potential role for PcrV in cancer immunotherapy through the targeting of TAM plasticity.

Pseudomonas aeruginosa is a virulent bacterium that secretes a variety of virulence factors that aid in establishing infections in individuals. Allicin, derived from garlic, has been shown to inhibit virulence factor production and biofilm formation in P. aeruginosa. However, the mechanisms underlying the allicin-mediated regulation of P. aeruginosa virulence remain unclear. In this study, we investigated the possible mechanisms underlying allicin-mediated virulence regulation in P. aeruginosa. The results showed that allicin attenuates the production of P. aeruginosa virulence-associated factors, such as elastase, pyocyanin, pyoverdine, and rhamnolipids, by inhibiting the rhl and pqs quorum-sensing systems. Further analysis revealed that the rhl and pqs systems play different roles during the allicin-mediated regulation process. Taken together, these results support the potential use of allicin as a therapeutic agent in controlling P. aeruginosa infection and associated mechanisms.

Coxsackievirus B3 (CVB3) is the predominant pathogen of viral myocarditis. In our previous study, we found that CVB3 caused abnormal lipid accumulation in host cells. However, the underlying mechanisms by which CVB3 disrupts and exploits the host lipid metabolism are not well understood. Sterol regulatory element binding protein 1 (SREBP1) is the major transcriptional factor in lipogenic genes expression. In this study, we demonstrated that CVB3 infection and nonstructural 2A protein upregulated and activated SREBP1a at the transcriptional level. Deletion analysis of SREBP1a promoter revealed that two regions, –1821/–1490 and –312/+217, in this promoter were both required for its activation by 2A. These promoter regions possessed several binding motifs for transcription factor SP1. Next, we used SP1-specific small interfering RNAs (siRNAs) to confirm that SP1 might be the essential factor in SREBP1a upregulation by 2A. Furthermore, we showed that MEK/ERK pathway was involved in the activation of SREBP1a by 2A and that blocking this signaling pathway with the specific inhibitor U0126 attenuated SREBP1a activation and lipid accumulation by 2A. Finally, we showed that inhibition of SREBP1 with siRNAs attenuated lipid accumulation induced by CVB3 infection and reduced virus replication. Moreover, inhibition of the MEK/ERK pathway also led to reduction of SREBP1a activation, lipid accumulation, and virus replication during CVB3 infection. Taken together, these data demonstrate that CVB3 nonstructural 2A protein activates SREBP1a at the transcription level through a mechanism involving MEK/ERK signaling pathway and SP1 transcription factor, which promotes cellular lipid accumulation and benefits virus replication. IMPORTANCE Coxsackievirus B3 (CVB3) infection is the leading cause of viral myocarditis, but effective vaccines and antiviral therapies against CVB3 infection are still lacking. It is important to understand the precise interactions between host and virus for the rational design of effective therapies. During infection, CVB3 disrupts and exploits host lipid metabolism to promote excessive lipid accumulation, which benefits virus replication. SREBP1 is the master regulator of cellular lipid metabolism. Here, we report that one of the viral nonstructural proteins, 2A, upregulates and activates SREBP1a. Furthermore, we find that inhibition of SREBP1 decreases CVB3 virus replication. These results reveal the regulation of SREBP1a expression by 2A and the roles of SREBP1 in lipid accumulation and viral replication during CVB3 infection. Our findings provide a new insight into CVB3 host interactions and inform a potential novel therapeutic target for this important pathogen.

BACKGROUND At present, the treatment of coxsackievirus-induced myocarditis remains difficult. Berberine (BBR), an isoquinoline alkaloid isolated from traditional medicine herbs, exhibits significant anti-viral efficacy against various viruses. However, the underlying mechanism by which BBR controls CVB3 infection has not yet been reported. The purpose of this study was to investigate the anti-viral efficacy of BBR against CVB3 infection and its mechanism. MATERIAL AND METHODS In our experiments, the protein levels of VP1 and MAPKs signal pathway were measured by Western blot. The mRNA level of VP1 was measured by RT-PCR. The virus titers were determined by TCID50 assay. RESULTS We found that BBR treatment significantly decreased CVB3 replication in HeLa cells. In addition, the BBR treatment reduced the phosphorylation levels of JNK and p38 MAPK upon CVB3 infection in both HeLa cells and primary rat myocardial cells. CONCLUSIONS Taken together, these results suggest that BBR inhibits CVB3 replication through the suppression of JNK and p38 MAPK activation, shedding new light on the investigation of therapeutic strategies against CVB3-induced viral myocarditis.

Coxsackievirus and adenovirus receptors (CARs) are the common cellular receptors which mediate coxsackievirus or adenovirus infection. Receptor trap therapy, which uses soluble viral receptors to block the attachment and internalization of virus, has been developed for the inhibition of virus infection. In this study, we have constructed a pPIC3.5 K/CAR-Fc expression plasmid for the economical and scale-up production of CAR-Fc fusion protein in Pichia pastoris . The coding sequence of the fusion protein was optimized according to the host codon usage bias. The amount of the CAR-Fc protein to total cell protein was up to 10% by 1% methanol induction for 96 h and the purity was up to 96% after protein purification. Next, the virus pull-down assay demonstrated the binding activity of the CAR-Fc to coxsackievirus. The analyses of MTT assay, immunofluorescence staining and quantitative real-time PCR after virus neutralization assay revealed that CAR-Fc could significantly block coxsackievirus B3 infection in vitro . In coxsackievirus B3 infected mouse models, CAR-Fc treatment reduced mortality, myocardial edema, viral loads and inflammation, suggesting the significant virus blocking effect in vivo . Our results indicated that the P. pastoris expression system could be used to produce large quantities of bioactive CAR-Fc for further clinical purpose.

Pathogenic bacteria could adjust gene expression to enable their survival in the distinct host environment. However, the mechanism by which bacteria adapt to the host environment is not well described. In this study, we demonstrated that nucleoside diphosphate kinase (Ndk) of Pseudomonas aeruginosa is critical for adjusting the bacterial virulence determinants during infection. Ndk expression was down-regulated in the pulmonary alveoli of a mouse model of acute pneumonia. Knockout of ndk up-regulated transcription factor ExsA-mediated T3S regulon expression and decreased exoproduct-related gene expression through the inhibition of the quorum sensing hierarchy. Moreover, in vitro and in vivo studies demonstrated that the ndk mutant exhibits enhanced cytotoxicity and host pathogenicity by increasing T3SS proteins. Taken together, our data reveal that ndk is a critical novel host-responsive gene required for coordinating P. aeruginosa virulence upon acute infection.

During infection, bacteria might generate adaptive responses to facilitate their survival and colonization in the host environment. The alarmone guanosine 5'-triphosphate-3'-diphosphate (ppGpp), the levels of which are regulated by the RelA and SpoT enzymes, plays a critical role in mediating bacterial adaptive responses and virulence. However, the mechanism by which ppGpp regulates virulence-associated traits in Pseudomonas aeruginosa is poorly understood. To investigate the regulatory role of ppGpp, the ppGpp-deficient strain ΔRS (relA and spoT gene double mutant) and the complemented strain ΔRS(++) (complemented with relA and spoT genes) were constructed. Herein, we reported that the ΔRS strain showed decreased cytotoxicity towards A549 human alveolar adenocarcinoma cell lines and led to reduced mortality, lung edema and inflammatory cell infiltration in a mouse model of acute pneumonia compared to wild-type PAO1 and the complemented strain ΔRS(++). Subsequent analyses demonstrated that the ΔRS strain displayed reduced T3SS expression, decreased levels of elastase activity, pyocyanin, pyoverdin and alginate, and inhibited swarming and biofilm formation compared to PAO1 and the complemented strain ΔRS(++). In addition, the results demonstrate that ppGpp-mediated regulation of T3SS, virulence factor production, and swarming occurs in a quinolone quorum-sensing system-dependent manner. Taken together, these results suggest that ppGpp is required for virulence regulation in P. aeruginosa, providing new clues for the development of interference strategies against bacterial infection.