VscF in T3SS1 Helps to Translocate VPA0226 in Vibrio parahaemolyticus

In Vibrio parahaemolyticus, type III secretion system 1 (T3SS1) is a major virulence factor that delivers effectors into the host eukaryotic cytoplasm; however, studies on its infection mechanism are currently limited. To determine the function of the vscF gene, we constructed the vscF deletion mutant ΔvscF and complementation strain CΔvscF. Compared with those of wild-type POR-1 and CΔvscF, the cytotoxic, adherent, and apoptotic abilities of ΔvscF in HeLa cells were significantly reduced (P < 0.01). Furthermore, in infected HeLa cells, the mutant strain reduced the translocation rates of VP1683 and VP1686 effectors compared to the wild-type and complementation strains. A BLAST search showed that vscF is homologous to the MixH needle protein of Shigella flexneri, indicating that the vscF gene encodes the needle protein of T3SS1 in V. parahaemolyticus. Additional translocation assays showed that VPA0226 translocated into the HeLa eukaryotic cytoplasm via T3SS1, secretion assays showed that VPA0226 can be secreted to supernatant by T3SS1, indicating that VPA0226 belongs to the unpublished class of T3SS1 effectors. In conclusion, our data indicate an essential role of vscF in V. parahaemolyticus T3SS1 and revealed that VPA0226 can be secreted into the host cell cytoplasm via T3SS1. This study provides insights into a previously unexplored aspect of T3SS1, which is expected to contribute to the understanding of its infection mechanism.

A Novel LysR Family Factor STM0859 is Associated with The Responses of Salmonella Typhimurium to Environmental Stress and Biofilm Formation

Salmonella enterica subsp. enterica serovar Typhimurium (ST) is an intracellularly parasitic bacterium. This zoonotic pathogen causes food poisoning and thus imposes a severe threat to food safety. Here, to understand the regulatory roles of the novel transcription factor STM0859 on the response of ST to environmental stress and biofilm formation, the STM0859 gene-deficient strain and the complementation strain ΔSTM0859/STM0859 were generated, respectively. Then, its capacity of responding to environmental stresses and biofilm (BF) formation ability under different stresses, including acid, alkali, high salt, cholate, and oxidative stresses was tested. We further analyzed the interaction between the STM0859 protein and the promoter of the acid stress response-related gene rcsB by performing an electrophoresis mobility shift assay (EMSA). The results showed that acid resistance and BF formation capacities of ST-ΔSTM0859 strain were significantly weaker, as compared with those of Salmonella Typhimurium SL1344 (ST-SL1344) wild strain (p < 0.01). Quantitative qRT-PCR analysis showed that the expression levels of acid stress and BF formation-related genes, rcsB and rpoS, of ST-ΔSTM0859 strain were significantly reduced at the transcription levels, while the transcription levels of these genes were fully restored in complementation strain ST-ΔSTM0859/STM0859. The results of EMSA showed that STM0859 was capable of binding the promoter DNA fragments of the rcsB gene, suggesting that STM0859 can promote the transcription of the rcsB gene through interaction with its promoter, thereby exerting an indirectly regulatory role on the adaptive responses to acid stress and BF formation of ST. This study provided new insights into the regulatory mechanisms of the LysR family factors on the tolerances of ST under adverse environmental stresses.

The Secreted Protein MoHrip1 Is Necessary for the Virulence of Magnaporthe oryzae

Secreted effectors from Magnaporthe oryzae play critical roles in the interaction with rice to facilitate fungal infection and disease development. M. oryzae-secreted protein MoHrip1 can improve plant defense as an elicitor in vitro, however, its biological function in fungal infection is not clear. In this study, we found that the expression of mohrip1 was significantly induced in the stages of fungal penetration and colonization. Although dispensable for the growth and conidiation, MoHrip1 was necessary for the full virulence of M. oryzae. Deletion of mohrip1 remarkably compromised fungal virulence on rice seedlings and even on rice leaves with wounds. Rice sheath inoculation assay further demonstrated the defects of mohrip1-deleted mutants on penetration and proliferation in rice cells. Additionally, compared with WT and complementation strain, the inoculation of mohrip1-deleted mutants induced a higher expression of specific defense related genes and a higher production of specific defensive compounds in rice leaves. These data collectively indicated that MoHrip1 is necessary for fungal penetration and invasive expansion, and further full virulence of rice blast fungus.

Novel Activator of Mannose-Specific Phosphotransferase System Permease Expression in Listeria innocua, Identified by Screening for Pediocin AcH Resistance

ABSTRACT To identify genes that are important for class IIa bacteriocin interaction and resistance in Listeria species, transposon Tn917 knockout libraries were constructed for Listeria innocua strain Lin11 and screened for mutants that are resistant to pediocin AcH. A highly resistant mutant (G7) (MIC > 20 μg/ml; 1,000-fold less susceptible than the wild type), in which the transposon integrated into the putative promoter of the lin0142 gene, was isolated. lin0142 is located immediately upstream of the mpt operon (mptA/mptC/mptD) that encodes the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system permease EIIt Man, which serves as a docking protein for class IIa bacteriocins. The transcription of the mpt operon is known to be positively controlled by σ54 factor and ManR (a σ54-associated activator). Transcripts for lin0142 and mpt were undetectable in the G7 mutant, based on quantitative real-time reverse transcriptase PCR analysis. When the wild-type lin0142 gene was expressed at a 7.9-fold-elevated level in the mutant via a multicopy-number plasmid, the level of mpt mRNA became 70% higher than that in the wild-type strain. In addition, the complementation strain reverted back to the pediocin AcH-susceptible phenotype. The levels of manR and rpoN (σ54) mRNAs were not directly influenced by the level of lin0142 transcription. lin0142 is the only one of the three mpt regulatory genes whose transcription is induced, albeit slightly (1.2-fold), by glucose. The combined results show that the lin0142 gene encodes a novel activator of the mpt operon. The Lin0142 protein contains a winged-helix DNA-binding motif and is distantly related to the Crp-Fnr family of transcription regulators.