LygA retention on the surface of Listeria monocytogenes via its interaction with wall teichoic acid modulates bacterial homeostasis and virulence.

Wall teichoic acid (WTA) is the abundant cell wall-associated glycopolymer in Gram-positive bacteria, playing crucial roles in surface proteins retention, bacterial homeostasis, and virulence. The WTA glycosylation of Listeria monocytogenes is essential for surface anchoring of virulence factors, whereas the nature and function of the noncovalent interactions between cell wall-associated proteins and WTA are less unknown. In this study, we found that galactosylated WTA (Gal-WTA) of serovar (SV) 4h L. monocytogenes plays a key role in modulating the novel glycine-tryptophan (GW) domain-containing autolysin protein LygA through direct interactions. Gal-deficient WTA of Lm XYSN (ΔgalT) showed a dramatic reduction of LygA on the cell surface. We demonstrated that LygA binds to Gal-WTA through the GW domains, and the binding affinity is associated with the number of GW motifs. Moreover, we confirmed the direct Gal-dependent binding of the GW protein Auto from the type I WTA strain, which has no interaction with rhamnosylated WTA, indicating that the complexity of both WTA and GW proteins affect the coordination patterns. Importantly, we revealed the crucial roles of LygA in facilitating bacterial homeostasis as well as crossing the intestinal and blood-brain barriers. Altogether, our findings suggest that both the glycosylation patterns of WTA and a fixed numbers of GW domains are closely associated with the retention of LygA on the cell surface, which promotes the pathogenesis of L. monocytogenes within the host.

A live attenuated DIVA vaccine affords protection against Listeria monocytogenes challenge in sheep.

Listeria monocytogenes (Lm) is a deadly foodborne pathogen that comprises 14 serotypes, among which, serotype 4b Lm is the primary cause of listeriosis outbreaks in humans and animals. Here, we evaluated the safety, immunogenicity, and protective efficacy of a serotype 4b vaccine candidate Lm NTSNΔactA/plcB/orfX in sheep. The infection dynamics, clinical features, and pathological observation verified that the triple genes deletion strain has adequate safety for sheep. Moreover, NTSNΔactA/plcB/orfX significantly stimulated humoral immune response and provided 78% immune protection to sheep against lethal wild-type strain challenge. Notably, the attenuated vaccine candidate could differentiate infected and vaccinated animals (DIVA) via serology determination of the antibody against listeriolysin O (LLO, encoded by hly) and phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB). These data suggest that the serotype 4b vaccine candidate has high efficacy, safety, and DIVA characteristics, and may be used to prevent Lm infection in sheep. Our study provides a theoretical basis for its future application in livestock and poultry breeding.

Discovery of novel DprE1 inhibitors via computational bioactivity fingerprints and structure-based virtual screening.

Decaprenylphosphoryl-ß-D-ribose oxidase (DprE1) plays important roles in the biosynthesis of mycobacterium cell wall. DprE1 inhibitors have shown great potentials in the development of new regimens for tuberculosis (TB) treatment. In this study, an integrated molecular modeling strategy, which combined computational bioactivity fingerprints and structure-based virtual screening, was employed to identify potential DprE1 inhibitors. Two lead compounds (B2 and H3) that could inhibit DprE1 and thus kill Mycobacterium smegmatis in vitro were identified. Moreover, compound H3 showed potent inhibitory activity against Mycobacterium tuberculosis in vitro (MICMtb = 1.25 µM) and low cytotoxicity against mouse embryo fibroblast NIH-3T3 cells. Our research provided an effective strategy to discover novel anti-TB lead compounds.

Epidemiology of Human Listeriosis in China During 2008-2017.

Listeria monocytogenes is a facultative, intracellular foodborne pathogen that causes listeriosis and is prevalent worldwide. However, our knowledge of this bacterium and the listeriosis it causes is still extremely limited until now. Therefore, this retrospective study of patients in mainland China over 10 years (2008-2017) was performed to better understand the demographic trends and clinical features of listeriosis in China. Both electronic and manual retrieval systems were used to collect the relevant literature on listeriosis in mainland China. A total of 759 cases were reported from 22 provinces. Among the clinical cases, septicemia was the most common presentation (49%), followed by central nervous system infection (25%). The overall case fatality rate was 18%, with a higher rate among neonatal patients (73%). In recent years, listeriosis has been reported annually and even peaked in 2014. The median age of nonperinatal cases was 36 years (range, 0-102), with a predominance of male cases (52%). Sporadic cases were frequent from March to May. Efforts to prevent and control the spread of listeriosis are required through further research and collaborative efforts to improve the capacities of clinical diagnosis and treatment.

Genetic Diversity of Listeria monocytogenes Isolates from Invasive Listeriosis in China.

Listeria monocytogenes is a deadly foodborne pathogen, and infections can result in meningoencephalitis and sepsis with mortality rates of up to 30%. In this study, we performed comparative whole-genome analysis of 30 clinical isolates sequenced together with 32 previously sequenced clinical and food isolates from China. The data indicate that L. monocytogenes isolates belonging to the clonal complexes (CC) -1, -8, -9, -87, -121, and -155 are present in human clinical cases. The majority of isolates are from CC-87, 9, and 8 and overlap with those CCs previously reported on the basis of multilocus sequence typing for isolates from Chinese food products. Detailed genome analysis of isolates, representative of CCs in clinical and food products, revealed strong similarities both in their core- and accessory genomes indicating that they are highly related. When compared to genome sequences of isolates of a given CC worldwide, clinical isolates of China were distinct and clustered in unified clades. Our data indicate that epidemic clones of L. monocytogenes (CC-87, 9, and 8) with unusually high occurrence of plasmids are unique to China and suggest that common populations of L. monocytogenes clones are present in both clinical and food products in China.

Rapid loss of early antigen-presenting activity of lymph node dendritic cells against Ag85A protein following Mycobacterium bovis BCG infection.

BACKGROUND: Control of Mycobacterium tuberculosis (Mtb) infection requires CD4+ T-cell responses and major histocompatibility complex class II (MHC II) presentation of Mtb antigens (Ags). Dendritic cells (DCs) are the most potent of the Ag-presenting cells and are central to the initiation of T-cell immune responses. Much research has indicated that DCs play an important role in anti-mycobacterial immune responses at early infection time points, but the kinetics of Ag presentation by these cells during these events are incompletely understood. RESULTS: In the present study, we evaluated in vivo dynamics of early Ag presentation by murine lymph-node (LN) DCs in response to Mycobacterium bovis bacillus Calmette-Guérin (BCG) Ag85A protein. Results showed that the early Ag-presenting activity of murine DCs induced by M. bovis BCG Ag85A protein in vivo was transient, appearing at 4 h and being barely detectable at 72 h. The transcription levels of CIITA, MHC II and the expression of MHC II molecule on the cell surface increased following BCG infection. Moreover, BCG was found to survive within the inguinal LN DC pool, representing a continuing source of mycobacterial Ag85A protein, with which LN DCs formed Ag85A peptide-MHCII complexes in vivo. CONCLUSIONS: Our results demonstrate that a decrease in Ag85A peptide production as a result of the inhibition of Ag processing to is largely responsible for the short duration of Ag presentation by LN DCs during BCG infection in vivo.

Expression of recombinant Newcastle disease virus F protein in Pichia pastoris and its immunogenicity using flagellin as the adjuvant.

Newcastle disease (ND), a highly contagious, acute, and potent infectious disease caused by Newcastle disease virus (NDV), has a considerable impact on the global poultry industry. Although both live attenuated and inactivated vaccines are used to prevent and control the spread of ND among chickens, the increasing number of ND outbreaks in commercial poultry flocks worldwide indicates that routine vaccinations are insufficient to control ND. Hence, efforts are being invested into developing alternative and more effective vaccination strategies. In this study, we focus on F protein, the neutralizing and protective antigen of NDV, and flagellin (FliC), a toll-like receptor 5 (TLR5) agonist that is an effective inducer of innate immune responses. We amplified F gene from velogenic NDV strain F48E8. The recombinant histidine (His)-tagged F protein was efficiently expressed in a Pichia pastoris (P. pastoris) eukaryotic system and verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The conditions for F protein expression in P. pastoris were optimal. The immunogenicity of F protein with FliC as the adjuvant was evaluated in a C3H/HeJ mouse model. FliC was found to enhance both F-specific and NDV-specific IgG responses and F-specific cellular immune responses following intraperitoneal co-administration with F protein. Thus, the recombinant F protein expressed by P. pastoris when used with flagellin as the adjuvant has potential as a subunit vaccine candidate.

[Expression and immunogenicity of Ag85A protein of Mycobacterium tuberculosis].

Objective: The aim of this study was to express Mycobacterium tuberculosis Ag85A protein and to evaluate its immunogenicity in mice. Methods: The cold expressed system and chaperone competent cells BL21 were combined to express Mycobacterium tuberculosis Ag85A protein, then the protein was purified with affinity chromatography and identified by Western Blot analysis. Results: The immunogenicity of the purified Ag85A protein was evaluated in C57BL/6 mice. Results show that high level of specific IgG was elicited in the serum, and the splenocytes and lymph node cells of immunized mice could produce more Th1 cytokines, such as IFN-γ and TNF-α, after stimulated with specific antigen. Conclusions: Ag85A protein can induce strong specific humoral and Th1 type cellular immune responses, providing an important biological material for further research and application.

Domain function dissection and catalytic properties of Listeria monocytogenes p60 protein with bacteriolytic activity.

The major extracellular protein p60 of Listeria monocytogenes (Lm-p60) is an autolysin that can hydrolyze the peptidoglycan of bacterial cell wall and has been shown to be required for L. monocytogenes virulence. The predicted three-dimensional structure of Lm-p60 showed that Lm-p60 could be split into two independent structural domains at the amino acid residue 270. Conserved motif analysis showed that V30, D207, S395, and H444 are the key amino acid residues of the corresponding motifs. However, not only the actual functions of these two domains but also the catalytic properties of Lm-p60 are unclear. We try to express recombinant Lm-p60 and identify the functions of two domains by residue substitution (V30A, D207A, S395A, and H444A) and peptide truncation. The C-terminal domain was identified as catalytic element and N-terminal domain as substrate recognition and binding element. Either N-terminal domain truncation or C-terminal domain truncation presents corresponding biological activity. The catalytic activity of Lm-p60 with a malfunctioned substrate-binding domain was decreased, while the substrate binding was not affected by a mulfunctioned catalytic domain. With turbidimetric method, we determined the optimal conditions for the bacteriolytic activity of Lm-p60 against Micrococcus lysodeikficus. The assay for the effect of Lm-p60 on the bacteriolytic activity of lysozyme revealed that the combined use of Lm-p60 protein with lysozyme showed a strong synergistic effect on the bacteriolytic activity.

Differential Effects of Mycobacterium bovis BCG on Macrophages and Dendritic Cells from Murine Spleen.

Macrophages (MΦ) and dendritic cells (DCs) are both pivotal antigen presenting cells capable of inducing specific cellular responses to inhaled mycobacteria, and thus, they may be important in the initiation of early immune responses to mycobacterial infection. In this study, we evaluated and compared the roles of murine splenic DCs and MΦs in immunity against Mycobacterium bovis Bacillus Calmette-Guérin (M.bovis BCG). The number of internalized rBCG-GFP observed was obviously greater in murine splenic MΦs compared with DCs, and the intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels in MΦs were all higher than in DCs. DCs have a stronger capacity for presenting Ag85A peptide to specific T hybridoma and when the murine splenic MΦs were infected with BCG and rBCG::Ag85A, low level of antigen presenting activity was detected. These data suggest that murine splenic MΦs participate in mycobacteria uptake, killing and inducing inflammatory response, whereas the murine splenic DCs are primarily involved in specific antigen presentation and T cell activation.

[Construction and characterization of Listeria monocytogenes deltahfq mutant strain].

OBJECTIVE: Gram-positive brevibacterium Listeria monocytogenes (Lm) is an important zoonotic foodborne pathogen, engaged in both saprophytism and parasitism. It could adapt, survive and display pathogenicity under different environmental stress challenges, which is associated with the regulatory network consisting of regulating factors. The biological characterizations of regulator hfq was evaluated in this study. METHODS: hfq deleted serovar 1/2 a strain EGDe was constructed with homologous recombination, the biological characteristics of the mutant strain was compared with its parental strain. RESULTS: The growth of EGDe delta hfq was significantly inhibited under cold temperature (P < 0.05), salt medium containing 7% NaCl and the medium containing 4.5% ethanol. The ability of biofilm formation of the mutant strain in BactoTM Brain Heart Infusion (BHI) was reduced significantly (P < 0.05); notably, the invasion rate to Caco-2 cell lines was obviously reduced. Infection capacity of EGDe delta hfq to BALB /c mice decreased and the LDD was 6 times higher than EGDe. CONCLUSION: Hfq protein of Listeria monocytogenes plays an important role in regulating bacterial virulence, biofilm formation and stress response. This deletion strain provided material to further study the function of Hfq and provides the possibilities to elucidate the mechanisms of Lm in resisting the stress and paves ways to the development of novel strategies for the prevention and control of Lm infections.

[Prokaryotic expression and immunological characteristics of Mycobacterium tuberculosis Rv1886c].

OBJECTIVE: Ag85B (Rv1886c) is secreted during the early stages of infection by Mycobacterium tuberculosis. The purpose of this study was probed into the immune response against Ag85B in vivo. METHODS: Ag85B was prokaryotic expressed and identified, its immunological characteristics were evaluated with indirect-ELSIA, Sandwich-ELISA and RESULTS: Ag85B was mainly expressed in form of inclusion body enzyme-linked immunospot assay (ELISPOT). confirmed by SDS-PAGE. Western blot analysis shows that the fusion protein had good specific reaction with serum of tuberculosis patient and serum of mice immunized with LM-Ag85B. C57BL/6 mice were subcutaneously immunized with Ag85B, the production of IFN-gamma and IL-4 in the spleen cells was determined by Sandwich ELISA, the level of IFN-gamma was significantly higher than that of IL-4 (P < 0.001) in the Ag85B immunization group, it indicated the protein induced Th1-tendency immune responses. Furthermore, purified protein derivative (PPD) used as coating antigen, antibody titer against Ag85B in murine serum reached 1:6400, it was demonstrated that Ag85B could also induce humoral immune responses. Additionally, C57BL/6 mice were intravenously immunized with M. tb H37Rv and bacillus Calmette-Guérin (BCG) respectively for 42 days, M. tb H37Rv group intended to induce Ag85B specific Th1 type immune response, and its ability of eliciting cellular immunity was significantly stronger than BCG group (P < 0.001). CONCLUSION: Ag85B can affectively induce strongly Th1-tendency immune response and humoral response. Whereas, BCG prime vaccination only can elicit low levels of Ag85B(240-259) specific immune response. The study laid foundation for probing the pathogenic mechanism, the development of novel vaccine and the establishment of clinical diagnostic method.

[Comparative proteomics analysis of extracellular proteins from Listeria monocytogenes and its isogenic prfA deletion mutant].

OBJECTIVE: Positive regulatory factor A (PrfA) protein plays a key role in the pathogenicity of Listeria monocytogenes by regulating the expression of virulence genes. We studied the regulation functions of PrfA and its role in Listeria monocytogenes (Lm) virulence. METHOD: Extracellular proteins were obtained from the supernatants of parental strain LM4 and mutant strain LM4deltaprfA cultured in minimal medium. We used two-dimensional gel electrophoresis and matrix associated laser dissociation/ionization time of flight mass spectrometry (MALDI- TOF-MS) to analyze the differences of secreted proteins between LM4 and LM4deltaprfA. RESULTS: The electrophoresis results show that 31 different spots, 19 spots corresponding 12 proteins were identified by MALDI- TOF-MS. Some virulence related proteins were verified, such as InlC, ActA and LLO. Some new proteins that are regulated by PrfA include D-alanyl-D-alanine carboxypeptidase, dipeptide Glycine and Trytophan (GW) repeat-containing surface protein, transcriptional regulator and some hypothetical proteins with unknown functions. Real-time quantitative PCR was conducted to verify the proteomics results. The mRNA expression level of hly, actA and inlC gene was significantly reduced, and that of D-alanyl-D-alanine carboxypeptidase and GW repeat-containing surface protein's synthesis also had a reduction in LM4deltaprfA strain. CONCLUSION: PrfA plays key roles on the regulation of genes in LIPI- I and LIPI- II.

[Immunobiologic characteristics of a recombinant Listeria monocytogenes expressing Mycobacterium tuberculosis antigens].

OBJECTIVE: Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis complex. Hence, novel vaccines against TB are urgently needed and important to the public health. METHODS: Immunobiologic characteristics of a recombinant attenuated Listeria monocytogenes strain LMdeltahly: :Ag85b-esat-6 was evaluated. RESULTS: LMdeltahly: :Ag85b-esat-6 had lost the hemolytic activity. It was completely cleared from the livers and spleens of mice 5 days after inoculation via intravenous route. Furthermore, the LD50 of the recombinant strain increased by 4 Logs comparing to that of the parent strain. Histopathology reveals no obvious pathological changes following administration of the recombinant strain to mice, indicating its safety. In addition, the potential protective immune response was evaluated on C57BL/6 mice via intravenous immunization route. The results indicate that the antigen delivered by the recombination LM could induce Th1 type immune response and elicit strong cytotoxic lymphocyte effect against Ag85B-ESAT-6. CONCLUSION: Thus, LMdeltahly::Ag85b-esat-6 had high safety to mice, and could be used as a novel vaccines candidate for preventing tuberculosis.

[Characterization of the cellular immune response induced by Mycobacterium tuberculosis Rv2626c].

Nearly a quarter of the world's population is infected with Mycobacterium tuberculosis and remains long-term asymptomatic infection. Rv2626c is a latent infection-related protein regulated by DosR of M. tuberculosis. In this study, the Rv2626c protein was prokaryotically expressed and purified, and its immunobiological characteristics were analyzed using RAW264.7 cells and mice as infection models. SDS-PAGE and Western blotting analysis showed that the Rv2626c-His fusion protein was mainly expressed in soluble form and specifically reacted with the rabbit anti-H37RV polyclonal serum. In addition, we found that the Rv2626c protein bound to the surface of RAW264.7 macrophages and up-regulated the production of NO. Moreover, the Rv2626c protein significantly induced the production of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6 and MCP-1, and induced strong Th1-tendency immune response. These results may help to reveal the pathogenic mechanism of M. tuberculosis and facilitate the development of new tuberculosis vaccine.

[Protective immune responses induced by a recombinant Listeria monocytogenes delivering HPV16 E7].

OBJECTIVE: To investigate specific immune responses elicited by a recombinant Listeria monocytogenes strain LM4 deltahly::E7 and assess protective effect in C57BL/6 mice. METHODS: C57BL/6 mice were intraperitoneally immunized with LM4 deltahly:: E7 at 1-week intervals. After the second immunization, cellular immunity elicited by this recombinant L. monocytogenes strain was analyzed via an ELISPOT assay, Cytotoxic T lymphocytes (CTL) measurement assay and analysis of effector T cells proportion in the splenocytes. Also, the serum antibodies against HPV16 E7 protein were determined in an ELISA assay. Finally, protective effect was assessed against the challenge with TC-1 tumor cells. RESULTS: The immune responses elicited by LM4 deltahly::E7 were biased towards Th1 type in the ELISPOT assay. Also, LM4 deltahly::E7 was able to induce E7-specific CTL activity, with average specific lysis of 72%, which was highly significant difference compared with the controls (P<0.01). Moreover, the proportion of effector T cells in the spleens were increased in mice immunized with recombinant L. monocytogenes strain (P<0.05). The titer of E7-specific antibodies in mice immunized with LM4 delta hly::E7 was 1:400 in the ELISA assay. Furthermore, immunization with LM deltahly::E7 protected all mice against the lethal tumor cell challenge. CONCLUSION: The data suggest that attenuated Listeria monocytogenes delivering HPV16 E7 antigen could induce both E7-specific cell mediated immunity and humoral immunity, and had a protective effect against challenge with TC-1 tumor cells.

New Insights into the Mechanism of Trichoderma virens-Induced Developmental Effects on Agrostis stolonifera Disease Resistance against Dollar Spot Infection.

Trichoderma is internationally recognized as a biocontrol fungus for its broad-spectrum antimicrobial activity. Intriguingly, the crosstalk mechanism between the plant and Trichoderma is dynamic, depending on the Trichoderma strains and the plant species. In our previous study, the Trichoderma virens 192-45 strain showed better pathogen inhibition through the secretive non-volatile and volatile substrates. Therefore, we studied transcriptional and metabolic responses altered in creeping bentgrass (Agrostis stolonifera L.) with T. virens colonization prior to a challenge with Clarireedia homoeocarpa. This fungal pathogen causes dollar spot on various turfgrasses. When the pathogen is deficient, the importance of T. virens to the enhancement of plant growth can be seen in hormonal production and microbe signaling, such as indole-3-acrylic acid. Therefore, these substrates secreted by T. virens and induced genes related to plant growth can be the 'pre-defense' for ensuing pathogen attacks. During C. homoeocarpa infection, the Trichoderma-plant interaction activates defense responses through the SA- and/or JA-dependent pathway, induced by T. virens and its respective exudates, such as oleic, citric, and stearic acid. Thus, we will anticipate a combination of genetic engineering and exogenous application targeting these genes and metabolites, which could make creeping bentgrass more resistant to dollar spot and other pathogens.

Tracking Clostridium perfringens strains from breeding duck farm to commercial meat duck farm by multilocus sequence typing.

Clostridium perfringens (C. perfringens) is an important zoonotic food-borne pathogenic microorganism. Currently, there are many reports on the prevalence of C. perfringens in poultry farms, while few studies on the prevalence and infection source of C. perfringens in egg hatcheries. The present study was undertaken to investigate and track C. perfringens from one breeding duck farm, one duck egg hatchery and one commercial meat duck farm along the production chain. A total of 334 isolates were obtained from 788 samples, including 316 type A strains (94.61 %), 11 type F strains (3.29 %) and seven type G strains (2.10 %). Antimicrobial susceptibility testing revealed that 61.87 % of the tested isolates were multidrug-resistant. Multilocus sequence typing showed that 66 representative isolates encompassed 60 different sequence types (STs), clustered in ten clonal complexes (CCs) and 20 singletons. CC2 was the most popular CC, accounting for 58.82 % (10/17) of the strains from breeding duck farm. Some strains of duck embryos were distributed in the same ST or CC as strains from breeding duck farm and duck egg hatchery, indicating close genetic relationship between them. The study showed that isolates from breeding duck farm had formed a dominant CC through long-term evolution, some duck embryos had been infected with C. perfringens during the incubation period and the infected strains should be from the duck egg hatchery or breeding duck farm with the possibility of vertical transmission. The close relationship between strains from breeding ducks and duck embryos, the high antibiotic resistance of isolates and the presence of cpe-positive or netB-positive isolates indicated that alert should be paid to such risks associated with these.

Transmembrane Protein LMxysn_1693 of Serovar 4h Listeria monocytogenes Is Associated with Bile Salt Resistance and Intestinal Colonization.

Listeria monocytogenes (Lm) is a ubiquitous foodborne pathogen comprising of 14 serotypes, of which serovar 4h isolates belonging to hybrid sub-lineage Ⅱ exhibit hypervirulent features. LMxysn_1693 of serovar 4h Lm XYSN, a member of genomic island-7 (GI-7), is predicted to a membrane protein with unknown function, which is conserved in serovar 4h Listeria monocytogenes. Under bile salts stress, Lm XYSN strain lacking LMxysn_1693 (XYSN∆1693) exhibited a stationary phase growth defect as well as a reduction in biofilm formation and strikingly down-regulated bile-salts-resistant genes and virulent genes. Particularly, LMxysn_1693 protein plays a crucial role in Lm XYSN adhesion and invasion to intestinal epithelial cells, as well as colonization in the ileum of mice. Taken together, these findings indicate that the LMxysn_1693 gene encodes a component of the putative ABC transporter system, synthetically interacts with genes involved in bile resistance, biofilm formation and virulence, and thus contributes to Listeria monocytogenes survival within and outside the host.

[Construction and characterization of Listeria monocytogenes deltaprfA mutant strains].

OBJECTIVE: Listeria monocytogenes (Lm) is an important pathogen that can cause serious listeriosis in humans and animals. The pathogenicity of Lm has a close relationship with the PrfA protein regulating the expression of virulence genes. Therefore, we studied the regulation functions of PrfA and its role on Lm's virulence. METHODS: The prfA genes of LM4, serotype 1/2a, and F4636, serotype 4b, were deleted by homologous recombination technology, and the biological characteristics of the mutants were further studied. RESULTS: The prfA gene deleted strains LM4deltaprfA and F4636deltaprfA and their back mutation strains were successfully constructed. The results show that the hemolysis activity was lost in prfA deleted strains and was recovered in the reverse mutant strains. The prfA deleted strains lost phospholipase activity; their adhesion and invasion ability significantly decreased. Furthermore, their 50% lethal doses (LD50) were 5 logs higher comparing with wild type strains. CONCLUSION: PrfA regulates hly, plcB and inl gene family and affects significantly Lm's virulence.