Avirulent Strain Research Articles

The Zymoseptoria tritici Avirulence Factor AvrStb6 Accumulates in Hyphae Close to Stomata and Triggers a Wheat Defense Response Hindering Fungal Penetration.

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is one of Europe's most damaging wheat pathogens, causing significant economic losses. Genetic resistance is a common strategy to control the disease, Stb6 being a resistance gene used for more than 100 years in Europe. This study investigates the molecular mechanisms underlying Stb6-mediated resistance. Utilizing confocal microscopy imaging, we determined that Z.tritici epiphytic hyphae mainly accumulate the corresponding avirulence factor AvrStb6 in close proximity to stomata. Consequently, the progression of AvrStb6-expressing avirulent strains is hampered during penetration. The fungal growth inhibition co-occurs with a transcriptional reprogramming in wheat characterized by an induction of immune responses, genes involved in stomatal regulation, and cell wall-related genes. Overall, we shed light on the gene-for-gene resistance mechanisms in the wheat-Z. tritici pathosystem at the cytological and transcriptomic level, and our results highlight that stomatal penetration is a critical process for pathogenicity and resistance. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Chitosan-Deficient Cryptococcus as Whole-Cell Vaccines.

Creating a safe and effective vaccine against infection by the fungal pathogen Cryptococcus neoformans is an appealing option that complements the discovery of new small molecule antifungals. Recent animal studies have yielded promising results for a variety of vaccines that include live-attenuated and heat-killed whole-cell vaccines, as well as subunit vaccines formulated around recombinant proteins. Some of the recombinantly engineered cryptococcal mutants in the chitosan biosynthesis pathway are avirulent and very effective at conferring protective immunity. Mice vaccinated with these avirulent chitosan-deficient strains are protected from a lethal pulmonary infection with C. neoformans strain KN99. Heat-killed derivatives of the vaccination strains are likewise effective in a murine model of infection. The efficacy of these whole-cell vaccines, however, is dependent on a number of factors, including the inoculation dose, route of vaccination, frequency of vaccination, and the specific mouse strain used in the study. Here, we present detailed methods for identifying and optimizing various factors influencing vaccine potency and efficacy in various inbred mouse strains using a chitosan-deficient cda1Δcda2Δcda3Δ strain as a whole-cell vaccine candidate. This chapter describes the protocols for immunizing three different laboratory mouse strains with vaccination regimens that use intranasal, orotracheal, and subcutaneous vaccination routes after the animals were sedated using two different types of anesthesia.

Trifluoroacetic acid-mediated synthesis of xanthene constructs and their extensive anti-tuberculosis evaluation.

A diverse range of 9-substituted 1,8-dioxohexahydroxanthenes was conceptualized and synthesized through a TFA-mediated approach in near quantitative yields without the use of column chromatography. From a series of 25 compounds, we found that compounds 14c and 14r exhibited promising anti-tuberculosis potential against avirulent and virulent strains of Mycobacterium tuberculosis with a Minimal Inhibitory Concentration (MIC) of 8 μg ml-1, achieving 99% bactericidal activity at the same concentration. This series of compounds was found to be inactive against common Gram-positive and Gram-negative pathogens, indicating that the activity is mycobacteria-specific. Since the strategies for treating tuberculosis employ a combinatorial therapy, we tested and observed that the two lead compounds displayed synergistic behavior with known anti-TB drugs (ATDs) and a significant (16-32 fold) decrease in MIC values of both leads was observed in combination with either RIF or INH. Interestingly the lead molecule 14c displayed only time-dependent kill kinetics and sterilized the whole culture of Mycobacterium tuberculosis H37Rv in just 48 hours.

Identification of Differential Circular RNA Expression Profiles and Functional Networks in Human Macrophages Induced by Virulent and Avirulent Mycobacterium tuberculosis Strains.

Circular RNAs (circRNAs) are noncoding RNAs with diverse functions. However, most Mycobacterium tuberculosis (M.tb)-related circRNAs remain undiscovered. In this study, we infected THP-1 cells with virulent and avirulent M.tb strains and then sequenced the cellular circRNAs. Bioinformatic analysis predicted 58,009 circRNAs in all the cells. In total, 2035 differentially expressed circRNAs were identified between the M.tb-infected and uninfected THP-1 cells and 1258 circRNAs were identified in the virulent and avirulent M.tb strains. Further, the top 10 circRNAs were confirmed by Sanger sequencing, among which four circRNAs, namely circSOD2, circCHSY1, circTNFRSF21, and circDHTKD1, which were highly differentially expressed in infected cells compared with those in uninfected cells, were further confirmed by ring formation, specific primers, and RNase R digestion. Next, circRNA-miRNA-mRNA subnetworks were constructed, such as circDHTKD1/miR-660-3p/IL-12B axis. Some of the individual downstream genes, such as miR-660-3p and IL-12B, were previously reported to be associated with cellular defense against pathological processes induced by M.tb infection. Because macrophages are important immune cells and the major host cells of M.tb, these findings provide novel ideas for exploring the M.tb pathogenesis and host defense by focusing on the regulation of circRNAs during M.tb infection.

Temporal Profiling of Host Proteome against Different M. tuberculosis Strains Reveals Delayed Epigenetic Orchestration.

Apart from being preventable and treatable, tuberculosis is the deadliest bacterial disease afflicting humankind owing to its ability to evade host defence responses, many of which are controlled by epigenetic mechanisms. Here, we report the temporal dynamics of the proteome of macrophage-like host cells after infecting them for 6, 18, 30, and 42 h with two laboratory strains (H37Ra and H37Rv) and two clinical strains (BND433 and JAL2287) of Mycobacterium tuberculosis (MTB). Using SWATH-MS, the proteins characterized at the onset of infection broadly represented oxidative stress and cell cytoskeleton processes. Intermediary and later stages of infection are accompanied by a reshaping of the combination of proteins implicated in histone stability, gene expression, and protein trafficking. This study provides strain-specific and time-specific variations in the proteome of the host, which might further the development of host-directed therapeutics and diagnostic tools against the pathogen. Also, our findings accentuate the importance of proteomic tools in delineating the complex recalibration of the host defence enabled as an effect of MTB infection. To the best of our knowledge, this is the first comprehensive proteomic account of the host response to avirulent and virulent strains of MTB at different time periods of the life span of macrophage-like cells. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD022352.

O-acetyl-homoserine sulfhydrylase deficient Streptococcus suis serotype 2 strain SC19 becomes an avirulent strain and provides immune protection against homotype infection in mice

O-acetyl-homoserine sulfhydrylase (OAHS) is a pyridoxal 5’-phosphate-dependent enzyme involved in microbial methionine biosynthesis, which catalyzes the conversion of o-acetyl-homoserine (OAH) to homocysteine. In our previous study, we found that OAHS of Streptococcus suis serotype 2 (SS2) can interact with the porcine blood-brain barrier (BBB) model, but whether OAHS regulates the penetration of BBB during SS2 infection is still unclear. To explore the role of OAHS in SS2 infection, OAHS-deficient SS2 mutant strain (SC19-ΔOAHS) and gene complemental strain (SC19-cΔOAHS) were constructed. Compared to the parent strain, with the loss of oahs, the chain length of SC19-ΔOAHS was shortened, the virulence was significantly reduced, the survival rate of mice infected with SC19-ΔOAHS was obviously increased accompanied by the relieved clinical symptoms. And the survival ability of SC19-ΔOAHS in whole blood was also remarkably decreased. Interestingly, the adhesion of SC19-ΔOAHS to endothelial cells was markedly increased, but the deficiency of OAHS significantly inhibited the strain penetrating BBB both in vivo and in vitro. Most of these phenomena can be reversed by the complemental strain (SC19-cΔOAHS). Further study showed that the deficiency of OAHS severely reduced SC19-induced endothelial cell apoptosis, tight junctions (TJs) protein impairment and the expression of SS2 virulence factor Enolase (Eno), involved in the destruction of BBB. Additionally, SC19-ΔOAHS immunized mice were able to resist SC19 or JZLQ022 infection. In conclusion, we confirmed that OAHS promoted the pathogenicity by enhancing host’s BBB permeability and immune escape, and SC19- ΔOAHS is a potential live vaccine.

T-independent B-cell effect of agents associated with swine grower-finisher diarrhea.

Swine dysentery, spirochetal colitis, and salmonellosis are production-limiting enteric diseases of global importance to the swine industry. Despite decades of efforts, mitigation of these diseases still relies on antibiotic therapy. A common knowledge gap among the 3 agents is the early B-cell response to infection in pigs. Thus, this study aimed to characterize the porcine B-cell response to Brachyspira hyodysenteriae, Brachyspira hampsonii (virulent and avirulent strains), Brachyspira pilosicoli, and Salmonella Typhimurium, the agents of the syndromes mentioned above. Immortalized porcine B-cell line derived from a crossbred pig with lymphoma were co-incubated for 8h with each pathogen, as well as E. coli lipopolysaccharide (LPS) and a sham-inoculum (n = 3/treatment). B-cell viability following treatments was evaluated using trypan blue, and the expression levels of B-cell activation-related genes was profiled using reverse transcription quantitative PCR. Only S. Typhimurium and LPS led to increased B-cell mortality. B. pilosicoli downregulated B-lymphocyte antigen (CD19), spleen associated tyrosine Kinase (syk), tyrosine-protein kinase (lyn), and Tumour Necrosis Factor alpha (TNF-α), and elicited no change in immunoglobulin-associated beta (CD79b) and swine leukocyte antigen class II (SLA-DRA) expression levels, when compared to the sham-inoculated group. In contrast, all other treatments significantly upregulated CD79b and stimulated responses in other B-cell downstream genes. These findings suggest that B. pilosicoli does not elicit an immediate T-independent B-cell response, nor does it trigger antigen-presenting mechanisms. All other agents activated at least one trigger within the T-independent pathways, as well as peptide antigen presenting mechanisms. Future research is warranted to verify these findings in vivo.

Using a dynamical model to study the impact of a toxoid vaccine on the evolution of a bacterium: The example of diphtheria

Vaccines are one of humankind's greatest weapons against infectious diseases. However, their usefulness is often questioned and the public tends to distrust vaccines. A mathematical model published in the early 2000s predicts the selection of more virulent strains of pathogens when populations are protected with imperfect vaccines, i.e., vaccines which reduce but do not entirely block pathogen transmission, such as toxoid vaccines. In this study, we built a disease-specific competition model to analyze the evolution of diphtheria's virulence under the pressure of a toxoid vaccine. Our results show that i) high vaccine coverage favors the emergence and increase prevalence of avirulent (or less virulent) strains of Corynebacterium diphtheriae (the etiologic agent of diphtheria) and ii) that competition between strains is crucial in the eradication of toxigenic strains when toxoid vaccines are used. We conclude that the use of toxoid vaccines could lead to disease eradication if the interaction between strains is taken into account. Our results could extend to biologically similar systems such as pertussis.

Evaluating the UV-C sensitivity of Coxiella burnetii in skim milk using a bench-scale collimated beam system and comparative thermal sensitivity study by high-temperature short-time pasteurization

Introduction:Coxiella burnetii is a zoonotic Gram-negative obligate intracellular bacterial pathogen and the causative agent of query (Q) fever in humans. Contamination of milk by C. burnetii, as a consequence of livestock infection, is a significant public health concern. Effective methods to inactivate C. burnetii in milk are a critical aspect of food safety. Implementation of non-thermal UV-C processing technologies in the dairy industry can effectively preserve the sensory and nutritional quality of raw milk products while ensuring their safety, making them a viable alternative to traditional high-temperature short-time (HTST) pasteurization methods.Methods: Optical light attenuation factors, such as the absorption, scattering, and reflection by skim milk (SM) were evaluated using a spectrophotometer. SM inoculated with an avirulent strain of C. burnetii was irradiated using a collimated beam device equipped with a low-pressure UV-C 254 nm lamp at doses from 0 to 12 mJ/cm2. Optical properties were considered for the evaluation of the delivered UV-C dose. The pasteurization treatment was conducted using a lab scale HTST pasteurizer (72°C/15 s). The verification studies were conducted using Escherichia coli ATCC 25922 inoculated in a phosphate buffer (transparent fluid) and humic acid (opaque fluid). Salmonella enterica serovar Muenchen ATCC BAA 1674 inoculated in SM was tested for its suitability as a surrogate for C. burnetii, a bacterium that requires specialized equipment and expertise for experimentation.Results and Discussion: Absorption, reduced scattering coefficient, and the reflectance of SM at 254 nm were measured as 19 ± 0.3/cm, 26 ± 0.5/cm, and 10.6%, respectively. The UV-C results showed a log-linear inactivation of C. burnetii in SM with the UV-C sensitivity (D10) value of 4.1 ± 0.04 mJ/cm2. The results of HTST pasteurization revealed that C. burnetii was heat-sensitive with a D value of 1.75 min. Salmonella Muenchen showed similar UV inactivation kinetics and is, thereby, suggested as a suitable surrogate to C. burnetii for the pilot-scale UV-C processing studies of SM.

An Avirulent Largemouth Bass Birnavirus Vaccine Candidate Protects Largemouth Bass against Birnavirus Infection.

Largemouth bass birnavirus (LBBV) disease outbreaks in largemouth bass fingerlings lead to high mortality in China. Therefore, the development of immersion immunization strategies is paramount. An avirulent LBBV strain was screened using a fish challenge assay. The proliferation dynamics of the avirulent strain were determined in vitro and in vivo. The efficacy of the avirulent vaccine was evaluated using immune gene expression, viral load, and a virus challenge, and the safety was also assessed using a reversion to virulence test. An avirulent virus strain, designated as largemouth bass birnavirus Guangdong Sanshui (LBBV-GDSS-20180701), was selected from five fish birnavirus isolates. The proliferation peak titer was 109.01 TCID50/mL at 24 hpi in CPB cells and the peak viral load was 2.5 × 104 copies/mg at 4 dpi in the head kidneys and spleens of largemouth bass. The largemouth bass that were immersed within an avirulent vaccine or injected with an inactivated vaccine were protected from the virulent LBBV challenge with a relative percent survival (RPS) of 75% or 42.9%, respectively. The expression levels of IL-12, MHCI, MHCII, CD8, CD4, and IgM in the avirulent group were significantly upregulated at a partial time point compared to the inactivated vaccine group. Moreover, the viral load in the avirulent vaccine group was significantly lower than those in the inactivated vaccine group and control group using real-time PCR. LBBV-GDSS-20180701 is a potential live vaccine candidate against LBBV disease.

Arabidopsis PHYTOALEXIN DEFICIENT 4 promotes the maturation and nuclear accumulation of immune-related cysteine protease RD19.

Arabidopsis PHYTOALEXIN DEFICIENT 4 (PAD4) has an essential role in pathogen resistance as a heterodimer with ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1). Here we investigated an additional PAD4 role in which it associates with and promotes the maturation of the immune-related cysteine protease RESPONSIVE TO DEHYDRATION 19 (RD19). We found that RD19 and its paralog RD19c promoted EDS1- and PAD4-mediated effector-triggered immunity to an avirulent Pseudomonas syringae strain, DC3000, expressing the effector AvrRps4 and basal immunity against the fungal pathogen Golovinomyces cichoracearum. Overexpression of RD19, but not RD19 protease-inactive catalytic mutants, in Arabidopsis transgenic lines caused EDS1- and PAD4-dependent autoimmunity and enhanced pathogen resistance. In these lines, RD19 maturation to a pro-form required its catalytic residues, suggesting that RD19 undergoes auto-processing. In transient assays, PAD4 interacted preferentially with the RD19 pro-protease and promoted its nuclear accumulation in leaf cells. Our results lead us to propose a model for PAD4-stimulated defense potentiation. PAD4 promotes maturation and nuclear accumulation of processed RD19, and RD19 then stimulates EDS1-PAD4 dimer activity to confer pathogen resistance. This study highlights potentially important additional PAD4 functions that eventually converge on canonical EDS1-PAD4 dimer signaling in plant immunity.

Virulent strains of Zymoseptoria tritici suppress the host immune response and facilitate the success of avirulent strains in mixed infections

Plants interact with a plethora of pathogenic microorganisms in nature. Pathogen-plant interaction experiments focus mainly on single-strain infections, typically ignoring the complexity of multi-strain infections even though mixed infections are common and critical for the infection outcome. The wheat pathogen Zymoseptoria tritici forms highly diverse fungal populations in which several pathogen strains often colonize the same leaf. Despite the importance of mixed infections, the mechanisms governing interactions between a mixture of pathogen strains within a plant host remain largely unexplored. Here we demonstrate that avirulent pathogen strains benefit from being in mixed infections with virulent strains. We show that virulent strains suppress the wheat immune response, allowing avirulent strains to colonize the apoplast and to reproduce. Our experiments indicate that virulent strains in mixed infections can suppress the plant immune system, probably facilitating the persistence of avirulent pathogen strains in fields planted with resistant host plants.

Comparative proteomics analysis of kidney in chicken infected by infectious bronchitis virus

The gamma coronavirus infectious bronchitis virus (IBV) is known to cause an acute and highly contagious infectious disease in poultry. Here, this study aimed to investigate the impact of virulent or avirulent IBV infection on the avian host by conducting proteomics with data-independent acquisition mass spectrometry (DIA-MS) in the kidneys of IBV-infected chickens. The results revealed 267, 489, and 510 differentially expressed proteins (DEPs) in the chicken kidneys at 3, 5, and 7 days postinfection (dpi), respectively, when infected with the GD17/04 strain, which is a highly nephrogenic strain and belongs to the 4/91 genotype. In contrast, the attenuated 4/91 vaccine resulted in the identification of 144, 175, and 258 DEPs at 3, 5, and 7 dpi, respectively. Functional enrichment analyses indicated distinct expression profiles between the 2 IBV strains. Upon GD17/04 infection, metabolic pathways respond initially in the early stage (3 dpi) and immune-related signaling pathways respond in the middle and late stages (5 and 7 dpi). The 4/91 vaccine elicited a completely opposite response compared to the GD17/04 infection. Among all DEPs, 62 immune-related DEPs were focused on and found to be mainly enriched in the type I interferon (IFN-I) signaling pathway and involved in humoral and cellular immunity. Notably, key molecules in the IFN-I signaling pathway including MDA5, LGP2, and TBK1 may serve as regulatory targets of IBV. Overall, this study highlights similarities and discrepancies in the patterns of protein expression at different stages of infection with virulent and avirulent IBV strains, with the IFN-I signaling pathway emerging as a critical response to IBV infection.

Testing a biocontrol agent consortium for suppression of tomato bacterial wilt through rhizosphere microecological regulation

Tomato bacterial wilt (TBW) caused by Ralstonia solanacearum leads to severe crop losses. The aim of the present study was to construct a biocontrol agent consortium (BAC) and evaluate its efficiency against TBW. Bacillus tequilensis FJAT-51047 and Streptomyces fradiae FJAT-31535 were isolated from healthy tomato rhizosphere soils and had intense antagonistic ability against R. solanacearum and high control efficiency against TBW. The mixture of fermentation broths of FJAT-1458 (an avirulent strain of R. solanacearum), FJAT-51047 and FJAT-31535 with a ratio (v:v:v) of 1:1:1 had the highest control efficiency of 100 % in a pot experiment and was used as the BAC. When it was applied in a 6-year continuous cropping field, the BAC could (1) enhance soil organic carbon, total nitrogen and phosphorus, and total potassium contents by 34.18, 38.46, 115.44, and 17.41 %, respectively, at the reproductive stage; (2) increase soil urease activity by 36.54 and 9.63 % at the vegetative and reproductive stages, respectively; (3) increase tomato yield by 113.46 %; and (4) reach a control efficiency of 84.10 %. The BAC significantly enriched rhizosphere soil with the beneficial bacteria Bauldia, Altererythrobacter, Pseudolabrys, Ferruginibacter and Rhodoplanes, and inhibited the pathogen-related bacteria Ralstonia and Aquicella. Redundancy analysis revealed a negative relationship between rhizosphere soil physicochemical parameters (pH, SOC, TN, TP and TK) and TBW disease incidence (DI) or the R. solanacearum population. Overall, BAC could be used for microecological regulation of the tomato rhizosphere to control TBW.

LysM Proteins TaCEBiP and TaLYK5 are Involved in Immune Responses Mediated by Chitin Coreceptor TaCERK1 in Wheat.

Plant lysin motif (LysM) ectodomain receptors interact with pathogen-associated molecular patterns (PAMPs) and have critical functions in plant-microbe interactions. In this study, 65 LysM family genes were identified using the recent version of the reference sequence of bread wheat (Triticum aestivum), in which 23, 16, 20, and 6 members belonged to LysM-containing receptor-like kinases (LYKs), LysM-containing receptor-like proteins (LYPs), extracellular LysM proteins (LysMes), and intracellular nonsecretory LysM proteins (LysMns), respectively. The study found that TaCEBiP, TaLYK5, and TaCERK1 were highly responsive to PAMP elicitors and phytopathogens, with TaCEBiP and TaLYK5 binding directly to chitin. TaCERK1 acted as a coreceptor with TaCEBiP and TaLYK5 at the plasma membrane. Overexpression of TaCEBiP, TaLYK5, and TaCERK1 in Nicotiana benthamiana leaves exhibited enhanced resistance to Sclerotinia sclerotiorum. Subsequently, knocking down TaCEBiP, TaLYK5, and TaCERK1 genes with barley stripe mosaic virus-VIGS compromised the wheat defense response to an avirulent strain of Puccinia striiformis. The study concluded that wheat has two synergistic chitin perception systems for detecting pathogen elicitors, with the activated CERK1 intracellular kinase domain leading to signaling transduction. This research provides valuable insights into the functional roles and regulatory mechanisms of wheat LysM members under biotic stress.

Susceptibility to Cryptococcus neoformans Infection with Bruton's Tyrosine Kinase Inhibition.

Patients receiving the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib have an increased likelihood of fungal infections. The objectives of this study were to determine if Cryptococcus neoformans infection severity was isolate dependent with BTK inhibition and whether blocking BTK impacted infection severity in a mouse model. We compared four clinical isolates from patients on ibrutinib to virulent (H99) and avirulent (A1-35-8) reference strains. BTK knockout (KO) and wild-type (WT) C57 mice and WT CD1 mice were infected by intranasal (i.n.), oropharyngeal aspiration (OPA), and intravenous (i.v.) routes. Infection severity was assessed by survival and fungal burden (CFU per gram of tissue). Ibrutinib (25 mg/kg) or vehicle was administered daily through intraperitoneal injections. In the BTK KO model, no isolate-dependent effect on fungal burden was observed, and infection severity was not significantly different from that of the WT with i.n., OPA, and i.v. routes. Ibrutinib treatment did not impact infection severity. However, when the four clinical isolates were compared to H99, two of these isolates were less virulent, with significantly longer survival and reduced rates of brain infection. In conclusion, C. neoformans infection severity in the BTK KO model does not appear to be isolate dependent. BTK KO and ibrutinib treatment did not result in significantly different infection severities. However, based on repeated clinical observations of increased susceptibility to fungal infections with BTK inhibitor therapy, further work is needed to optimize a mouse model with BTK inhibition to better understand the role that this pathway plays in susceptibility to C. neoformans infection.

Cooperation between physiological defenses and immune resistance produces asymptomatic carriage of a lethal bacterial pathogen.

Animals evolved two defense strategies to survive infections. Antagonistic strategies include immune resistance mechanisms that operate to kill invading pathogens. Cooperative or physiological defenses mediate host adaptation to the infected state, limiting physiological damage and disease, without killing the pathogen, and have been shown to cause asymptomatic carriage and transmission of lethal pathogens. Here, we demonstrate that physiological defenses cooperate with the adaptive immune response to generate long-term asymptomatic carriage of the lethal enteric murine pathogen, Citrobacter rodentium. Asymptomatic carriage of genetically virulent C. rodentium provided immune resistance against subsequent infections. Immune protection was dependent on systemic antibody responses and pathogen virulence behavior rather than the recognition of specific virulent antigens. Last, we demonstrate that an avirulent strain of C. rodentium in the field has background mutations in genes that are important for LPS structure. Our work reveals insight into how asymptomatic infections can arise mechanistically with immune resistance, mediating exclusion of phenotypically virulent enteric pathogen to promote asymptomatic carriage.

A live attenuated RHΔompdcΔuprt mutant of Toxoplasma gondii induces strong protective immunity against toxoplasmosis in mice and cats

BackgroundToxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis. It is essential to develop an effective anti-T. gondii vaccine for the control of toxoplasmosis, and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats.MethodsFirst, the ompdc and uprt genes of T. gondii were deleted through the CRISPR-Cas9 system. Then, the intracellular proliferation and virulence of this mutant strain were evaluated. Subsequently, the immune responses induced by this mutant in mice and cats were detected, including antibody titers, cytokine levels, and subsets of T lymphocytes. Finally, the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats. Furthermore, to discover the effective immune element against toxoplasmosis, passive immunizations were carried out. GraphPad Prism software was used to conduct the log-rank (Mantel–Cox) test, Student’s t test and one-way ANOVA.ResultsThe RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system. Compared with the wild-type strain, the mutant notably reduced proliferation (P < 0.05). In addition, the mutant exhibited virulence attenuation in both murine (BALB/c and BALB/c-nu) and cat models. Notably, limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice. Furthermore, compared with nonimmunized group, high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (IFN-γ, IL-4, IL-10, IL-2 and IL-12) in mice were detected by the mutant (P < 0.05). Remarkably, all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains. The immunized sera and splenocytes, especially CD8+ T cells, could significantly extend (P < 0.05) the survival time of mice challenged with the RHΔku80 strain compared with naïve mice. In addition, compared with nonimmunized cats, cats immunized with the mutant produced high levels of antibodies and cytokines (P < 0.05), and notably decreased the shedding numbers of oocysts in feces (95.3%).ConclusionsThe avirulent RHΔompdcΔuprt strain can provide strong anti-T. gondii immune responses, and is a promising candidate for developing a safe and effective live attenuated vaccine.Graphical abstract

Increased Production of Pathogenic, Airborne Fungal Spores upon Exposure of a Soil Mycobiota to Chlorinated Aromatic Hydrocarbon Pollutants.

Organic pollutants are omnipresent and can penetrate all environmental niches. We evaluated the hypothesis that short-term (acute) exposure to aromatic hydrocarbon pollutants could increase the potential for fungal virulence. Specifically, we analyzed whether pentachlorophenol and triclosan pollution results in the production of airborne fungal spores with greater virulence than those derived from an unpolluted (Control) condition. Each pollutant altered the composition of the community of airborne spores compared to the control, favoring an increase in strains with in vivo infection capacity (the wax moth Galleria mellonella was used as an infection model). Fungi subsisting inside larvae at 72 h postinjection with airborne spore inocula collected in polluted and unpolluted conditions exhibited comparable diversity (mainly within Aspergillus fumigatus). Several virulent Aspergillus strains were isolated from larvae infected with the airborne spores produced in a polluted environment. Meanwhile, strains isolated from larvae injected with spores from the control, including one A. fumigatus strain, showed no virulence. Potential pathogenicity increased when two Aspergillus virulent strains were assembled, suggesting the existence of synergisms that impact pathogenicity. None of the observed taxonomic or functional traits could separate the virulent from the avirulent strains. Our study emphasizes pollution stress as a possible driver of phenotypic adaptations that increase Aspergillus pathogenicity, as well as the need to better understand the interplay between pollution and fungal virulence. IMPORTANCE Fungi colonizing soil and organic pollutants often meet. The consequences of this encounter constitute an outstanding question. We scrutinized the potential for virulence of airborne fungal spores produced under unpolluted and polluted scenarios. The airborne spores showed increased diversity of strains with higher infection capacity in Galleria mellonella whenever pollution is present. Inside the larvae injected with either airborne spore community, the surviving fungi demonstrated a similar diversity, mainly within Aspergillus fumigatus. However, the isolated Aspergillus strains greatly differ since virulence was only observed for those associated with a polluted environment. The interplay between pollution and fungal virulence still hides many unresolved questions, but the encounter is costly: pollution stress promotes phenotypic adaptations that may increase Aspergillus pathogenicity.

Public Health Importance of Preventive Measures for Salmonella Tennessee and Salmonella Typhimurium Strain LT2 Biofilms

Various serovars of Salmonella had been the subject of research for over 150 years; nonetheless, the bacterium has remained an important pathogen of public health concern to date. The tremendous ability of Salmonella to form biofilms on biotic and abiotic surfaces is an important underlying reason for the prevalence of this opportunistic pathogen in healthcare, manufacturing, and the food chain. The current study illustrates that using very common industrial antimicrobial treatments at the highest concentrations suggested by the manufacturers is only efficacious against planktonic and one-day mature biofilms of the pathogen while exhibiting a lack of efficacy for complete removal of bacterial biofilms formed for longer than 2 days. This exhibits the importance of preventive measures against Salmonella biofilm formation in healthcare and manufacturing facilities, schools, nursing homes, and domestic environments. Additionally, our study illustrates the importance of including both planktonic and sessile cells of the pathogen in microbiology validation studies, especially for niche and hard-to-reach surfaces. The current study additionally investigated the suitability of an avirulent strain of the pathogen as a surrogate for pathogenic Salmonella serovars for public health microbiology validation studies when the use of virulent strains is not economically feasible or not possible due to safety concerns.