ABSTRACT
Listeria monocytogenes is a facultative anaerobic Gram-positive bacterium. It is well adapted to external environments and able to infect both humans and animals. To understand the impacts of ncRNA Rli60 on the adaptability of L. monocytogenes to environmental stresses and biofilm formation, a rli60 deletion strain of L. monocytogenes (LM-Δrli60) was constructed using splicing by overlap extension PCR (SOE-PCR) and homologous recombination and then compared it with wild-type strain L. monocytogenes EGD-e in the aspects of adaptability to environmental stresses by measuring their growth under stresses of different temperatures, and acidic, alkaline, hypertonic and alcoholic conditions, and capability of biofilm formation by using crystal violet staining, as well as the transcriptional levels of genes (gltB and gltC) related to the biofilm formation by real-time quantitative PCR (qRT-PCR). The results showed that (1) the growth of LM-Δrli60 strain was significantly slower under environmental stresses of low temperature (30 °C), high temperature (42 °C), as well as alkaline and alcoholic conditions, (2) the amount of biofilm formed by LM-Δrli60 was attenuated, and (3) the transcriptional levels of gltB and gltC genes at 24 h and 48 h in LM-Δrli60 revealed a significant reduction. Overall, the results confirmed that ncRNA Rli60 plays important roles in regulating the adaptability of L. monocytogenes to environmental stresses and biofilm formation possibly through impacting the expression of gltB and gltC genes.
Subject(s)
Biofilms , Gene Expression Regulation, Bacterial , Listeria monocytogenes/physiology , RNA, Bacterial/metabolism , RNA, Long Noncoding/metabolism , Adaptation, Physiological , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Humans , Listeria monocytogenes/genetics , RNA, Bacterial/genetics , RNA, Long Noncoding/genetics , Stress, PhysiologicalABSTRACT
BACKGROUND: Listeria monocytogenes (LM) is an important zoonotic foodborne pathogen. Noncoding RNA (ncRNA) has an important role in regulating its virulence. As a member of ncRNA, however, the function of Rli60 in regulating LM virulence remain unclear. The aim of this study was to investigate the role of Rli60 in regulating LM virulence. METHODS: Using a homologous recombination method, a LM EGD-e rli60 gene deletion strain (LM-Δrli60) was constructed and compared with a LM EGD-e strain in the following respects: (1) adhesiveness, invasion ability, intracellular survival, proliferation, and transcription of virulence genes in the mouse macrophage cell line RAW264.7; (2) 50% lethal dose (LD50) to the BALB/c mouse; and (3) the amount in the mouse liver and spleen and the effects on pathology of mouse liver, spleen, and kidney after inoculation. RESULTS: The LM-Δrli60 strain had a significantly higher adhesion rate and lower invasion rate with significantly lower intracellular survival and proliferation rates in the RAW264.7 cell line, compared to the LM EGD-e strain. Inoculation with LM-Δrli60 strain significantly affected the transcription of virulence genes. The LD50 of LM-Δrli60 to BALB/c mouse was increased by 2.12 logarithmic magnitude, which indicated that the virulence in LM-Δrli60 is significantly decreased (p < 0.05). The amount of LM-Δrli60 in the liver and spleen was significantly lower than the amount of LM EGD-e in these organs (p < 0.05). The pathological damage due to LM-Δrli60 infection in the mouse liver, spleen, and kidney was lower than the damage due to LM EGD-e infection. CONCLUSION: This study confirmed that the rli60 deletion could significantly affect LM virulence, adhesion, invasion, survival, and proliferation. This suggests that Rli60 has an important role in regulating LM virulence.
Subject(s)
Bacterial Adhesion/genetics , Kidney/microbiology , Listeria monocytogenes/genetics , Listeria monocytogenes/pathogenicity , Liver/microbiology , Spleen/microbiology , Animals , Cell Line , Gene Deletion , Kidney/pathology , Listeria monocytogenes/growth & development , Listeriosis/microbiology , Liver/pathology , Macrophages/microbiology , Mice , Mice, Inbred BALB C , RNA, Untranslated/genetics , Spleen/pathology , Virulence/geneticsABSTRACT
Orf is an exanthemous viral disease seriously threatening the goat and sheep industry and widely epidemic in the goat and sheep populations in Xinjiang, China. In order to investigate the genetic variability of the orf virus (ORFV), three virus isolates (SHZ1, SHZ2, and SHZ3) were isolated by PCR and Vero cell culture using the clinical samples from the lips of the lambs suspected of ORFV infection. The isolates were further verified by electron microscopy and animal infection experiments. The protective antigen genes B2L, F1L, and virulence genes VIR, GIF, and VEGF in the isolates were cloned, sequenced and analyzed for genetic evolution. The results showed that B2L and F1L were relatively conservative with homology 86.7-97.9%, while VIR, GIF, particularly VEGF were considerably variable with homology 71.5-97.9% at amino acid sequence level, respectively. Phylogenetic tree analysis based on B2L and VIR showed that the isolates SHZ1 and SHZ2 were closely related with the Taiwan isolates. This is the first report to confirm that there have been genetic variations in the Xinjiang ORFV isolates. The findings provide molecular evidence about the genetic variability of the major antigenic and virulence genes in the virus isolates epidemic in Xinjiang.
