Biology subjects

Huang, I.-H.

Publications and source records attributed to Huang, I.-H..

4 recordsLinked to original sources

bioRxiv2025

Characterization of Clinical Fusobacterium nucleatum Isolates from Oral Squamous Cell Carcinoma Patients

Fusobacterium nucleatum (Fn), a Gram-negative anaerobe primarily residing in the oral cavity, has garnered increasing attention for its role in a broad spectrum of human diseases. While typically absent or rarely detected outside the oral cavity in healthy individuals, Fn is frequently found at extra-oral sites under disease conditions and has been implicated in cancer progression and prognosis1. In oral squamous cell carcinoma (OSCC), the abundance of Fn significantly increases as the disease progresses, promoting cell invasion and metastasis. Furthermore, substantial evidence links Fn to accelerated tumor growth and metastatic progression in colorectal cancer (CRC), where its presence is also associated with chemotherapy resistance and poor prognosis. Here, to further elucidate the pathogenic mechanisms of Fn in cancer progression, this study characterized the physiological traits, virulence factor expression, and impacts on cancer cells of 10 Fn strains, including two well-characterized ATCC strains and eight clinical isolates. The clinical isolates consisted of three strains from saliva samples of 117 OSCC patients and five strains from 160 non-cancer individuals. Results indicated that oral isolates, regardless of disease origin, all belong to Fn subspecies polymorphum. The ATCC strains (23726 and 25586) exhibited shorter cell lengths and faster growth rates compared to the clinical isolates. Both ATCC strains formed stable biofilms and expressed key virulence genes, including aim1, fadA, fomA, and radD. All isolates tested showed sensitivity to a panel of eight different antibiotics. Interestingly, only one clinical isolate displayed similar stimulation of CRC cell migration as the two ATCC strains, while the other seven displayed no such ability. Collectively, these findings suggest that the most virulent strains, in terms of biofilm formation and virulence gene expression, are not necessarily the most pathogenic in the context of cancer cell interactions. Future pan-genomic analyses, incorporating whole-genome sequencing of the clinical isolates, will aim to delineate the genetic determinants contributing to the carcinogenic potential of Fn.

microbiology↗

bioRxiv2024

The protease interpain A of Prevotella intermedia promotes human OSCC cells proliferation and migration

Prevotella intermedia has been increasingly recognized as a potential contributor to oral squamous cell carcinoma (OSCC), yet the underlying mechanisms remain poorly defined. In this study, we identified interpain A (InpA), a cysteine protease secreted by P. intermedia, as a key virulence factor that promotes oral epithelial cell proliferation and OSCC cell migration. Conditioned medium (CM) derived from P. intermedia strain ATCC 25611 enhanced proliferation in both normal oral keratinocytes (SG cells) and OSCC cell lines (SCC-15, SAS). The pro-proliferative effect was abolished upon heat inactivation of the CM and inhibited by E64, a cysteine protease inhibitor, and FSLLRY-NH2, a PAR-2 antagonist, suggesting the involvement of protease-activated receptor-2 (PAR-2) signaling. InpA was highly secreted by strain ATCC 25611 but not by clinical isolates lacking proliferative effects, and RT-qPCR confirmed higher inpA expression in OSCC-derived strains compared to non-cancer controls. Recombinant InpA (rInpA) mimicked the effect of CM, inducing proliferation and migration, which were blocked by PAR-2 inhibition. Transcriptomic and protein-level screening in SG cells revealed activation of EGFR and downstream RAS-RAF-MEK-ERK signaling. Furthermore, in a colorectal cancer (CRC) mouse model, oral administration of P. intermedia led to increased tumor formation, suggesting a broader oncogenic potential. These findings highlight InpA as a PAR-2-activating protease that may contribute to OSCC and potentially other cancers associated with oral microbiota dysbiosis.

cancer biology↗

bioRxiv2024

Combination of mitomycin C and low-dose metronidazole synergistically against Clostridioides difficile infection and recurrence prevention

Clostridioides difficile is an anaerobic, spore-forming, Gram-positive pathogen responsible for various conditions from mild diarrhea to severe toxic megacolon and potentially death. Current treatments for C. difficile infection (CDI) rely on antibiotics like vancomycin and metronidazole (MTZ); however, the high doses required often disrupt gut microbiota, leading to a high recurrence rate. Mitomycin C (MMC), a chemotherapy drug approved by the FDA, is known for inducing phage production in lysogenic bacterial strains, effectively targeting the host bacteria. Given that 70% of C. difficile strains harbor prophages, this study investigates MMCs potential to enhance antibiotic efficacy against CDI. Our in vitro experiments indicate that MMC acts synergistically with MTZ to inhibit the growth of C. difficile strain R20291. Furthermore, this combination decreases biofilm-resident vegetative cell resistance and reduces the MTZ concentration needed to kill C. difficile in stool samples ex vivo. In a CDI mouse relapse model, in vivo results show that MMC combined with a low dose of MTZ significantly improves survival rates and reduces fecal spore counts after antibiotic treatment. Overall, these findings suggest that a low-dose combination of MMC and MTZ offers enhanced therapeutic efficacy for CDI, both in vitro and in vivo, and may provide a promising new approach for treatment.

microbiology↗

bioRxiv2023

Phage transcriptional regulator X (PtrX)-mediated augmentation of toxin production and virulence in Clostridioides difficile strain R20291

Clostridioides difficile is a Gram-positive, anaerobic, and spore-forming bacterial member of the human gut microbiome. The primary virulence factors of C. difficile are toxin A and toxin B. These toxins damage the cell cytoskeleton and cause various diseases, from diarrhea to severe pseudomembranous colitis. Evidence suggests that bacteriophages can regulate the expression of the pathogenic locus (PaLoc) genes of C. difficile. We previously demonstrated that the genome of the C. difficile strain RT027 (NCKUH-21) contains a prophage-like DNA sequence, which was found to be markedly similar to that of the {varphi}CD38-2 phage. In the present study, we investigated the mechanisms underlying the {varphi}NCKUH-21-mediated regulation of the pathogenicity and the PaLoc genes expression in the lysogenized C. difficile strain R20291. The carriage of {varphi}NCKUH-21 in R20291 cells substantially enhanced toxin production, bacterial motility, biofilm formation, and spore germination in vitro. Subsequent mouse studies revealed that the lysogenized R20291 strain caused a more severe infection than the wild-type strain. We screened three {varphi}NCKUH-21 genes encoding DNA-binding proteins to check their effects on PaLoc genes expression. The overexpression of NCKUH-21_03890, annotated as a transcriptional regulator (phage transcriptional regulator X, PtrX), considerably enhanced toxin production, biofilm formation, and bacterial motility of R20291. Transcriptome analysis further confirmed that the overexpression of ptrX led to the upregulation of the expression of toxin genes, flagellar genes, and csrA. In the ptrX-overexpressing R20291 strain, PtrX influenced the expression of flagellar genes and the sigma factor gene sigD, possibly through an increased flagellar phase ON configuration ratio. Author SummaryClostridioides difficile is a Gram-positive, spore-forming anaerobic bacterium that can lead to antibiotic-associated diarrhea and pseudomembranous colitis. During the C. difficile infection (CDI), the major virulence factor is the secretion of two exotoxins, toxin A and B, to destroy host intestinal epithelium cells. The investigation of bacteriophages affecting the toxicity of C. difficile has increasingly been research. We previously isolated a C. difficile clinical strain NCKUH-21, which carried a phage-like DNA sequence, and named it {varphi}NCKUH-21. However, whether this prophage could enhance the virulence of C. difficile and the mechanism for regulating the pathogenicity are still unclear. We successfully created a {varphi}NCKUH-21-lysogenized R20291 strain and showed that lysogenized R20291 performed stronger pathogenicity than the wild-type R20291. We found that a {varphi}NCKUH-21-specific protein (encoded by NCKUH-21_03890 gene) might influence C. difficile flagellar phase variation to promote toxin production further. These findings are expected to clarify the mechanism for controlling the pathogenicity of {varphi}NCKUH-21-infected C. difficile. Moreover, we also believe that the existence of hypervirulent C. difficile strains carrying a prophage should be monitored proactively in hospitals to prevent severe CDI.

microbiology↗