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1.
Molecules ; 26(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34443685

RESUMO

Recognition of pathogen-associated molecular patterns (PAMPs) by appropriate pattern recognition receptors (PRRs) is a key step in activating the host immune response. The role of a fungal PAMP is attributed to ß-1,3-glucan. The role of α-1,3-glucan, another fungal cell wall polysaccharide, in modulating the host immune response is not clear. This work investigates the potential of α-1,3-glucan as a fungal PAMP by analyzing the humoral immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan. We demonstrated that 57-kDa and 61-kDa hemolymph proteins, identified as ß-1,3-glucan recognition proteins, bound to A. niger α-1,3-glucan. Other hemolymph proteins, i.e., apolipophorin I, apolipophorin II, prophenoloxidase, phenoloxidase activating factor, arylphorin, and serine protease, were also identified among α-1,3-glucan-interacting proteins. In response to α-1,3-glucan, a 4.5-fold and 3-fold increase in the gene expression of antifungal peptides galiomicin and gallerimycin was demonstrated, respectively. The significant increase in the level of five defense peptides, including galiomicin, corresponded well with the highest antifungal activity in hemolymph. Our results indicate that A. niger α-1,3-glucan is recognized by the insect immune system, and immune response is triggered by this cell wall component. Thus, the role of a fungal PAMP for α-1,3-glucan can be postulated.


Assuntos
Aspergillus/química , Glucanos/metabolismo , Interações Hospedeiro-Patógeno , Mariposas/microbiologia , Padrões Moleculares Associados a Patógenos/metabolismo , Animais , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Corpo Adiposo/efeitos dos fármacos , Corpo Adiposo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hemolinfa/metabolismo , Imunização , Larva , Mariposas/efeitos dos fármacos , Mariposas/genética , Ligação Proteica/efeitos dos fármacos , Análise de Sobrevida
2.
J Med Microbiol ; 70(8)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34397349

RESUMO

Introduction. Lactococcus petauri LZys1 (L. petauri LZys1) is a type of lactic acid bacteria (LAB), which was initially isolated from healthy human gut.Hypothesis/Gap Statement. It was previously anticipated that L. petauri LZys1 has potential characteristics of probiotic properties. The genetic structure and the regulation functions of L. petauri LZys1 need to be better revealed.Aim. The aim of this study was to detect the probiotic properties L. petauri LZys1 and to reveal the genome information related to its genetic adaptation and probiotic profiles.Methodology. Multiple in vitro experiments were carried out to evaluate its lactic acid-producing ability, resistance to pathogenic bacterial strains, auto-aggregation and co-aggregation ability, and so on. Additionally, complete genome sequencing, gene annotation, and probiotic associated gene analysis were performed.Results. The complete genome of L. petauri LZys1 comprised of 1 985 765 bp, with a DNA G+C content of 38.07 %, containing 50 tRNA, seven rRNA, and four sRNA. A total of 1931 genes were classified into six functional categories by Kyoto Encyclopaedia of Genes and Genomes (KEGG) database. The neighbour-joining phylogeny tree based on the whole genome of L. petauri LZys1 and other probiotics demonstrated that L. petauri LZys1 has a significant similarity to Lactococcus garvieae. The functional genes were detected to expound the molecular mechanism and biochemical processes of its potential probiotic properties, such as atpB gene.Conclusion. All the results described in this study, together with relevant information previously reported, made L. prtauri LZys1 a very interesting potential strain to be considered as a prominent candidate for probiotic use.


Assuntos
Trato Gastrointestinal/microbiologia , Genoma Bacteriano , Lactococcus , Probióticos , Animais , Bactérias/crescimento & desenvolvimento , Bactérias/patogenicidade , Sequência de Bases , Fezes/microbiologia , Genes Bacterianos , Humanos , Lactococcus/citologia , Lactococcus/genética , Lactococcus/isolamento & purificação , Lactococcus/fisiologia , Masculino , Anotação de Sequência Molecular , Mariposas/microbiologia , Filogenia , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/genética , Sequenciamento Completo do Genoma , Adulto Jovem
3.
Neotrop Entomol ; 50(5): 804-811, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34398398

RESUMO

Bacillus thuringiensis (Bt)-based bioinsecticides and transgenic plants expressing proteins with insecticidal activity (Cry and Vip) have been successfully used in several integrated pest management programs worldwide. Lepidoptera comprise some of the most economically important insect pests of the major agricultural crops. In this study, the toxicity of 150 Bt strains was evaluated against Helicoverpa armigera (Hübner) larvae. Eight strains (426, 520B, 1636, 1641, 1644, 1648, 1657 and 1658) showed high insecticide activity against H. armigera and were therefore tested against Anticarsia gemmatalis (Hübner), Spodoptera cosmioides (Walker), Chrysodeixis includens (Walker), and Diatraea saccharalis (Fabricius) larvae. Our results showed that most of the Bt strains were also toxic to these lepidopteran species. The biochemical and molecular analyses of these strains revealed that they had a similar protein profile; however, their cry and vip gene contents were variable. In addition, the median lethal concentration (LC50) of the selected strains indicated that the strains 1636, 1641, and 1658 were the most effective against H. armigera, showing LC50 values of 185.02, 159.44, and 192.98 ng/cm2, respectively. Our results suggest that the selected Bt strains have great potential to control the lepidopteran pests H. armigera, A. gemmatalis, D. saccharalis, S. cosmioides, and C. includes.


Assuntos
Bacillus thuringiensis , Agentes de Controle Biológico , Mariposas , Controle Biológico de Vetores , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas/toxicidade , Proteínas Hemolisinas , Larva/microbiologia , Mariposas/microbiologia
4.
Nucleic Acids Res ; 49(12): 6925-6940, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34151378

RESUMO

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.


Assuntos
RNA Helicases DEAD-box/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Aclimatação , Adenosina Trifosfatases/metabolismo , Animais , Temperatura Baixa , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/classificação , RNA Helicases DEAD-box/fisiologia , Endorribonucleases/metabolismo , Mariposas/microbiologia , Filogenia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , RNA/metabolismo , Estabilidade de RNA , Análise de Sequência de RNA , Virulência
5.
Appl Environ Microbiol ; 87(17): e0074821, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34160271

RESUMO

Several fungi, including the plant root symbiont and insect pathogen Metarhizium brunneum, produce lysergic acid amides via a branch of the ergot alkaloid pathway. Lysergic acid amides include important pharmaceuticals and pharmaceutical lead compounds and have potential ecological significance, making knowledge of their biosynthesis relevant. Many steps in the biosynthesis of lysergic acid amides have been determined, but terminal steps in the synthesis of lysergic acid α-hydroxyethylamide (LAH)-by far the most abundant lysergic acid amide in M. brunneum-are unknown. Ergot alkaloid synthesis (eas) genes are clustered in the genomes of fungi that produce these compounds, and the eas clusters of LAH producers contain two uncharacterized genes (easO and easP) not found in fungi that do not produce LAH. Knockout of easO via a CRISPR-Cas9 approach eliminated LAH and resulted in accumulation of the alternate lysergic acid amides lysergyl-alanine and ergonovine. Despite the elimination of LAH, the total concentration of lysergic acid derivatives was not affected significantly by the mutation. Complementation with a wild-type allele of easO restored the ability to synthesize LAH. Substrate feeding studies indicated that neither lysergyl-alanine nor ergonovine were substrates for the product of easO (EasO). EasO had structural similarity to Baeyer-Villiger monooxygenases (BVMOs), and labeling studies with deuterated alanine supported a role for a BVMO in LAH biosynthesis. The easO knockout had reduced virulence to larvae of the insect Galleria mellonella, indicating that LAH contributes to virulence of M. brunneum on insects and that LAH has biological activities different from ergonovine and lysergyl-alanine. IMPORTANCE Fungi in the genus Metarhizium are important plant root symbionts and insect pathogens. They are formulated commercially to protect plants from insect pests. Several Metarhizium species, including M. brunneum, were recently shown to produce ergot alkaloids, a class of specialized metabolites studied extensively in other fungi because of their importance in agriculture and medicine. A biological role for ergot alkaloids in Metarhizium species had not been demonstrated previously. Moreover, the types of ergot alkaloids produced by Metarhizium species are lysergic acid amides, which have served directly or indirectly as important pharmaceutical compounds. The terminal steps in the synthesis of the most abundant lysergic acid amide in Metarhizium species and several other fungi (LAH) have not been determined. The results of this study demonstrate the role of a previously unstudied gene in LAH synthesis and indicate that LAH contributes to virulence of M. brunneum on insects.


Assuntos
Aminas/metabolismo , Proteínas Fúngicas/metabolismo , Ácido Lisérgico/metabolismo , Metarhizium/enzimologia , Oxigenases de Função Mista/metabolismo , Animais , Vias Biossintéticas , Proteínas Fúngicas/genética , Larva/microbiologia , Metarhizium/genética , Metarhizium/metabolismo , Metarhizium/patogenicidade , Oxigenases de Função Mista/genética , Mariposas/microbiologia , Virulência
6.
Infect Immun ; 89(7): e0057920, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-33875476

RESUMO

Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a noncanonical type VI secretion system (T6SS), in an arthropod in vivo infection model is missing. Here, we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. tularensis subsp. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of the T6SS effectors PdpC, PdpD, or OpiA is sufficient to kill G. mellonella larvae, while sheath recycling by ClpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK, OpiB1, OpiB2, and OpiB3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into the function of T6SS and pathogenesis of Francisella.


Assuntos
Proteínas de Bactérias/genética , Francisella tularensis/fisiologia , Larva/microbiologia , Mariposas/microbiologia , Sistemas de Secreção Tipo VI/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Francisella tularensis/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Tularemia , Sistemas de Secreção Tipo VI/efeitos dos fármacos , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
7.
Arch Microbiol ; 203(6): 3509-3517, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33909089

RESUMO

Klebsiella pneumoniae is an important human pathogen causing urinary tract infections and pneumonia. Due to the increase in resistant strains and being an opportunistic pathogen, it is very important to determine the virulence process, the cellular damage it causes in the host and the immunological response level of the host. In this study, invertebrate infection model Galleria mellonella larvae were used to investigate cellular damage, antioxidant response and changes in biochemical parameters due to K. pneumoniae infection. The activity of cell damage indicators alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase increased in hemolymph of G. mellonella larvae due to K. pneumoniae virulence. Creatine kinase, alkaline phosphatase, gamma glutamyl transferase and amylase activities were increased to regulate the disrupted energy metabolism due to infection. As a result of the damage caused by K. pneumoniae infection, changes occurred in the amount of non-enzymatic antioxidants, uric acid, bilirubin and albumin. Due to K. pneumoniae infection, the amount of calcium, potassium, magnesium and phosphorus altered. This study showed that G. mellonella larvae was important infection model in the investigation of infectious cell damage and physiological effects, given the opportunistic nature of the K. pneumoniae pathogen and the lack of adequate animal models.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Mariposas , Animais , Modelos Animais de Doenças , Infecções por Klebsiella/fisiopatologia , Klebsiella pneumoniae/fisiologia , Larva/microbiologia , Mariposas/microbiologia
8.
Res Vet Sci ; 136: 598-601, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33895568

RESUMO

Pseudomonas (P.) aeruginosa is the most frequently isolated Gram-negative bacteria in dog otitis. Antimicrobial resistance is particularly prevalent in P. aeruginosa and phage therapy represents a promising alternative therapeutic strategy. The aim of this study was to assess the efficacy of the PEV2 phage against a clinical P. aeruginosa isolate from a canine otitis using a Galleria (G.) mellonella larvae model. The genomic DNA of PAV237 P. aeruginosa isolate was sequenced and analysed. In a first main experiment, the efficacy of PEV2 phage against PAV237 was assessed at different multiplicities of infection (MOI) (50,000, 5000, 500, 50) by analyzing the larvae survival rate during 4 days. In a second experiment, the bacterial and phage titer evolutions were assessed depending on two MOIs (50,000, 5000). No significant survival increase was observed with PEV2 therapy in the infected larvae groups. The generated Kaplan-Meier curves showed that the rate of alive larvae was significantly higher in the non-infected larvae compared to the infected-treated ones irrespective of phage MOIs. An increase of the phage titer was observed at 24 and 48 h post-inoculation (HPI) with both MOIs and the P. aeruginosa titers were lower with MOI 50,000 and 5000 compared to the infectivity control at 24 and 48 HPI. Even if an ineffectiveness of the PEV2 phage was observed on the larvae survival, PEV2 is active against P. aeruginosa in this model and PEV2 replication is correlated with a lower bacterial proliferation in the phage treated larvae.


Assuntos
Doenças do Cão/terapia , Mariposas/microbiologia , Otite/veterinária , Terapia por Fagos/veterinária , Infecções por Pseudomonas/veterinária , Fagos de Pseudomonas , Pseudomonas aeruginosa/virologia , Animais , Cães , Larva/microbiologia , Otite/terapia , Infecções por Pseudomonas/terapia
9.
J Insect Physiol ; 131: 104239, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33845095

RESUMO

The work presents identification of antimicrobial peptides and proteins (AMPs) in the hemolymph of Galleria mellonella larvae infected with two Pseudomonas aeruginosa strains (ATCC 27,853 and PA18), differing in the profile of secreted proteases. The insects were immunized with bacteria cultivated in rich (LB) and minimal (M9) media, which resulted in appearance of a similar broad set of AMPs in the hemolymph. Among them, 13 peptides and proteins were identified, i.e. proline-rich peptides 1 and 2, lebocin-like anionic peptide 1 and anionic peptide 2, defensin/galiomicin, cecropin, cecropin D-like peptide, apolipophoricin, gallerimycin, moricin-like peptide B, lysozyme, apolipophorin III, and superoxide dismutase. Bacterial strain- and/or medium-dependent changes in the level of proline-rich peptide 1, anionic peptide 1 and 2, moricin-like peptide B, cecropin D-like and gallerimycin were observed. The analysis of the expression of genes encoding cecropin, gallerimycin, and galiomicin indicated that they were differently affected by the bacterial strain but mainly by the medium used for bacterial culture. The highest expression was found for the LB medium. In addition to the antibacterial and antifungal activity, proteolytic activity was detected in the hemolymph of the P. aeruginosa-infected insects. Based on these results and those presented in our previous reports, it can be postulated that the appearance of AMPs in G. mellonella hemolymph can be triggered not only by P. aeruginosa pathogen associated molecular patterns (PAMPs) but also by bacterial extracellular proteases secreted during infection. However, although there were no qualitative differences in the set of AMPs depending on the P. aeruginosa strain and medium, differences in the level of particular AMPs synthesized in response to the bacteria used were observed.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Interações Hospedeiro-Patógeno , Mariposas/metabolismo , Peptídeo Hidrolases/metabolismo , Pseudomonas aeruginosa/enzimologia , Animais , Hemolinfa/metabolismo , Larva/metabolismo , Larva/microbiologia , Mariposas/microbiologia
10.
mBio ; 12(2)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33906924

RESUMO

Amoeboid predators, such as amoebae, are proposed to select for survival traits in soil microbes such as Cryptococcus neoformans; these traits can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Consistent with this notion, incubation of various fungal species with amoebae enhanced their virulence, but the mechanisms involved are unknown. In this study, we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged times and then analyzed surviving colonies phenotypically and genetically. Surviving colonies comprised cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of traits associated with virulence, such as capsule size, urease production, and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding the oligopeptide transporter (CNAG_03013; OPT1) were observed among amoeba-passaged isolates from each of the three strains. Isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene encoding Pkr1 (AMP-dependent protein kinase regulator) but manifested reduced virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate that C. neoformans survival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes. Given the myriad potential predators in soils, the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation.IMPORTANCE Cryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for an animal host in its life cycle. Previous studies showed that C. neoformans increases its pathogenicity after interacting with its environmental predator amoebae. Amoebae, like macrophages, are phagocytic cells that are considered an environmental training ground for pathogens to resist macrophages, but the mechanism by which C. neoformans changes its virulence through interactions with protozoa is unknown. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival, with this diversity suggesting a bet-hedging strategy to ensure that some forms survive.


Assuntos
Acanthamoeba castellanii/fisiologia , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Fagocitose , Acanthamoeba castellanii/microbiologia , Animais , Criptococose/imunologia , Cryptococcus neoformans/classificação , Cryptococcus neoformans/genética , Citocinas/imunologia , Feminino , Humanos , Larva/microbiologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Mariposas/microbiologia , Fagócitos/microbiologia , Fenótipo , Virulência
11.
Toxins (Basel) ; 13(3)2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33809820

RESUMO

Concerns about resistance development to conventional insecticides in diamondback moth (DBM) Plutella xylostella (L.), the most destructive pest of Brassica vegetables, have stimulated interest in alternative pest management strategies. The toxicity of Bacillus thuringiensis subsp. aizawai (Bt GO33A) combined with chlorantraniliprole (Chl) has not been documented. Here, we examined single and combined toxicity of chlorantraniliprole and Bt to assess the levels of resistance in four DBM strains. Additionally, enzyme activities were tested in field-original highly resistant (FOH-DBM), Bt-resistant (Bt-DBM), chlorantraniliprole-resistant (CL-DBM), and Bt + chlorantraniliprole-resistant (BtC-DBM) strains. The Bt product had the highest toxicity to all four DBM strains followed by the mixture of insecticides (Bt + Chl) and chlorantraniliprole. Synergism between Bt and chlorantraniliprole was observed; the combination of Bt + (Bt + Chl) (1:1, LC50:LC50) was the most toxic, showing a synergistic effect against all four DBM strains with a poison ratio of 1.35, 1.29, 1.27, and 1.25. Glutathione S-transferase (GST) and carboxyl-esterase (CarE) activities showed positive correlations with chlorantraniliprole resistance, but no correlation was observed with resistance to Bt and Bt + Chl insecticides. Expression of genes coding for PxGST, CarE, AChE, and MFO using qRT-PCR showed that the PxGST and MFO were significantly overexpressed in Bt-DBM. However, AChE and CarE showed no difference in the four DBM strains. Mixtures of Bt with chlorantraniliprole exhibited synergistic effects and may aid the design of new combinations of pesticides to delay resistance in DBM strains substantially.


Assuntos
Bacillus thuringiensis/metabolismo , Brassica/parasitologia , Resistência a Inseticidas , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Mariposas/microbiologia , Controle Biológico de Vetores , ortoaminobenzoatos/farmacologia , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Animais , Bacillus thuringiensis/genética , Carboxilesterase/genética , Carboxilesterase/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Resistência a Inseticidas/genética , Mariposas/enzimologia , Mariposas/genética
12.
J Insect Physiol ; 131: 104213, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33662378

RESUMO

Some insects display immunological priming as a result of elevated humoral and cellular responses which give enhanced survival against subsequent infection. The humoral immune response of Galleria mellonella larvae following pre-exposure to heat killed Staphylococcus aureus or Candida albicans cells was determined by quantitative mass spectrometry in order to assess the relationship between the humoral immune response and resistance to subsequent bacterial or fungal infection. Larvae pre-exposed to heat killed S. aureus showed increased resistance to subsequent bacterial and fungal infection. Larvae displayed an increased hemocyte density (14.08 ± 2.14 × 106 larva-1 (p < 0.05) compared to the PBS injected control [10.41 ± 1.67 × 106 larva-1]) and increased abundance of antimicrobial proteins (cecropin-D-like peptide (+22.23 fold), hdd11 (+12.61 fold) and prophenol oxidase activating enzyme 3 (+5.96 fold) in response to heat killed S. aureus. Larvae pre-exposed to heat killed C. albicans cells were resistant to subsequent fungal infection but not bacterial infection and showed a reduced hemocyte density (6.01 ± 1.63 × 106 larva-1 (p < 0.01) and increased abundance of hdd11 (+32.73 fold) and moricin-like peptide C1 (+16.76 fold). While immune priming is well recognised in G. mellonella larvae the results presented here indicate distinct differences in the response of larvae following exposure to heat killed bacterial and fungal cells.


Assuntos
Interações Hospedeiro-Patógeno , Imunidade Celular , Imunidade Humoral , Larva/imunologia , Mariposas/imunologia , Animais , Candida albicans , Hemócitos , Larva/metabolismo , Larva/microbiologia , Mariposas/metabolismo , Mariposas/microbiologia , Proteoma , Staphylococcus aureus
13.
Virulence ; 12(1): 818-834, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33682618

RESUMO

The invertebrate Galleria mellonella has increasingly and widely been used in the last few years to study complex host-microbe interactions. Aspergillus fumigatus is one of the most pathogenic fungi causing life-threatening diseases in humans and animals. Galleria mellonella larvae has been proven as a reliable model for the analysis of pathogenesis and virulence factors, enable to screen a large number of A. fumigatus strains. This review describes the different uses of G. mellonella to study A. fumigatus and provides a comparison of the different protocols to trace fungal pathogenicity. The review also includes a summary of the diverse mutants tested in G. mellonella, and their respective contribution to A. fumigatus virulence. Previous investigations indicated that G. mellonella should be considered as an interesting tool even though a mammalian model may be required to complete and verify initial data.


Assuntos
Aspergilose/microbiologia , Aspergillus fumigatus/patogenicidade , Larva/microbiologia , Mariposas/microbiologia , Fatores de Virulência/análise , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/efeitos dos fármacos , Modelos Animais de Doenças , Farmacorresistência Fúngica , Interações Hospedeiro-Patógeno , Virulência
14.
Virulence ; 12(1): 638-653, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33550901

RESUMO

Adhesins facilitate bacterial colonization and invasion of host tissues and are considered virulence factors, but their impact on immune-mediated damage as a driver of pathogenesis remains unclear. Yersinia pseudotuberculosis encodes for a multivalent adhesion molecule (MAM), a mammalian cell entry (MCE) family protein and adhesin. MAMs are widespread in Gram-negative bacteria and enable enteric bacteria to colonize epithelial tissues. Their role in bacterial interactions with the host innate immune system and contribution to pathogenicity remains unclear. Here, we investigated howY. pseudotuberculosis MAM contributes to pathogenesis during infection of the Galleria mellonella insect model. We show that Y. pseudotuberculosis MAM is required for efficient bacterial binding and uptake by hemocytes, the host phagocytes. Y. pseudotuberculosis interactions with insect and mammalian phagocytes are determined by bacterial and host factors. Loss of MAM, and deficient microbe-phagocyte interaction, increased pathogenesis in G. mellonella. Diminished phagocyte association also led to increased bacterial clearance. Furthermore, Y. pseudotuberculosis that failed to engage phagocytes hyperactivated humoral immune responses, most notably melanin production. Despite clearing the pathogen, excessive melanization also increased phagocyte death and host mortality. Our findings provide a basis for further studies investigating how microbe- and host-factors integrate to drive pathogenesis in a tractable experimental system.


Assuntos
Interações Hospedeiro-Patógeno , Larva/microbiologia , Mariposas/microbiologia , Fagócitos/microbiologia , Fagócitos/patologia , Yersinia pseudotuberculosis/metabolismo , Adesinas Bacterianas , Animais , Hemócitos , Mariposas/citologia , Fagócitos/imunologia , Fatores de Virulência , Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/patogenicidade , Infecções por Yersinia pseudotuberculosis/microbiologia
15.
Arch Insect Biochem Physiol ; 106(3): e21769, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33590536

RESUMO

Upon entry into the hemocoel of host insects, entomopathogenic fungi switch to yeast-like hyphal bodies that are not recognized by host hemocytes and replicate extensively in the hemolymph. The mechanism by which hyphal bodies evade host cellular immunity is not well understood. This study compares Metarhizium rileyi conidia and hyphal bodies with respect to elicitation of the immune response of Helicoverpa armigera and recognition by host pattern recognition receptors (PRRs). We found that the ability of host hemocytes to phagocytize and nodulate hyphal bodies was weaker than those responses against conidia, suggesting that hyphal bodies are more able to evade host cellular immunity. Additionally, we found that the binding affinity of H. armigera ß-1,3-glucan recognition proteins was much lower for hyphal bodies than for conidia. We observed no agglutination response of H. armigera C-type lectin 3 (HaCTL3) against hyphal bodies, and HaCTL3 bound significantly less to hyphal bodies than to conidia, indicating that host PRRs have a lower affinity for hyphal bodies than for conidia. This study provides direct evidence that the mechanism whereby entomopathogenic fungi escape host cellular immunity involves the inability of host PRRs to sufficiently recognize hyphal bodies to elicit the cellular immune response.


Assuntos
Interações entre Hospedeiro e Microrganismos , Imunidade Celular , Metarhizium/imunologia , Mariposas/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Aglutinação/fisiologia , Animais , Hemócitos/metabolismo , Hemolinfa/citologia , Hemolinfa/metabolismo , Hifas/imunologia , Evasão da Resposta Imune , Lectinas Tipo C/metabolismo , Mariposas/microbiologia , Fagocitose , Esporos Fúngicos/imunologia
16.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33397694

RESUMO

Two FRQ proteins (Frq1 and Frq2) distinct in molecular mass and structure coexist in Beauveria bassiana, an asexual insect-pathogenic fungus. Frq1 and Frq2 have been proven to have opposite nuclear rhythms that can persistently activate developmental activator genes and hence orchestrate nonrhythmic conidiation in vitro under light or in darkness. Here, we report the essentiality of either FRQ, but Frq2 being more important than Frq1, for the fungal virulence and infection cycle. The fungal virulence was attenuated significantly more in the absence of frq2 than in the absence of frq1 through either normal cuticle infection or cuticle-bypassing infection by intrahemocoel injection, accompanied by differentially reduced secretion of Pr1 proteases required for the cuticle infection and delayed development of hyphal bodies in vivo, which usually propagate by yeast-like budding in the host hemocoel to accelerate insect death from mycosis. Despite insignificant changes in radial growth under normal, oxidative, and hyperosmotic culture conditions, conidial yields of the Δfrq1 and Δfrq2 mutants on insect cadavers were sharply reduced, and the reduction increased with shortening daylight length on day 9 or 12 after death, indicating that both Frq1 and Frq2 are required for the fungal infection cycle in host habitats. Intriguingly, the Δfrq1 and Δfrq2 mutants showed hypersensitivity and high resistance to cell wall-perturbing calcofluor white, coinciding respectively with the calcofluor-triggered cells' hypo- and hyperphosphorylated signals of Slt2, a mitogen-activated protein kinase (MAPK) required for mediation of cell wall integrity. This finding offers a novel insight into opposite roles of Frq1 and Frq2 in calcofluor-specific signal transduction via the fungal Slt2 cascade.IMPORTANCE Opposite nuclear rhythms of two distinct FRQ proteins (Frq1 and Frq2) coexisting in an asexual fungal insect pathogen have been shown to orchestrate the fungal nonrhythmic conidiation in vitro in a circadian day independent of photoperiod change. This paper reports essential roles of both Frq1 and Frq2, but a greater role for Frq2, in sustaining the fungal virulence and infection cycle since either frq1 or frq2 deletion led to marked delay of lethal action against a model insect and drastic reduction of conidial yield on insect cadavers. Moreover, the frq1 and frq2 mutants display hypersensitivity and high resistance to cell wall perturbation and have hypo- and hyperphosphorylated MAPK/Slt2 in calcofluor white-triggered cells, respectively. These findings uncover a requirement of Frq1 and Frq2 for the fungal infection cycle in host habitats and provide a novel insight into their opposite roles in calcofluor-specific signal transduction through the MAPK/Slt2 cascade.


Assuntos
Beauveria/metabolismo , Beauveria/patogenicidade , Proteínas Fúngicas/metabolismo , Mariposas/microbiologia , Virulência , Animais , Benzenossulfonatos , Larva/microbiologia , Transdução de Sinais
17.
Int J Antimicrob Agents ; 57(3): 106283, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33503451

RESUMO

A major determinant of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is the drug insensitive transpeptidase, PBP2a, encoded by mecA. Full expression of the resistance phenotype requires auxiliary factors. Two such factors, auxiliary factor A (auxA, SAUSA300_0980) and B (auxB, SAUSA300_1003), were identified in a screen against mutants with increased susceptibility to ß-lactams in the MRSA strain, JE2. auxA and auxB encode transmembrane proteins, with AuxA predicted to be a transporter. Inactivation of auxA or auxB enhanced ß-lactam susceptibility in community-, hospital- and livestock-associated MRSA strains without affecting PBP2a expression, peptidoglycan cross-linking or wall teichoic acid synthesis. Both mutants displayed increased susceptibility to inhibitors of lipoteichoic acid (LTA) synthesis and alanylation pathways and released LTA even in the absence of ß-lactams. The ß-lactam susceptibility of the aux mutants was suppressed by mutations inactivating gdpP, which was previously found to allow growth of mutants lacking the lipoteichoic synthase enzyme, LtaS. Using the Galleria mellonella infection model, enhanced survival of larvae inoculated with either auxA or auxB mutants was observed compared with the wild-type strain following treatment with amoxicillin. These results indicate that AuxA and AuxB are central for LTA stability and potential inhibitors can be tools to re-sensitize MRSA strains to ß-lactams and combat MRSA infections.


Assuntos
Antibacterianos/farmacologia , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/metabolismo , Ácidos Teicoicos/metabolismo , Amoxicilina/farmacologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cefoxitina/farmacologia , Parede Celular/metabolismo , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Humanos , Larva/microbiologia , Proteínas de Membrana/genética , Meropeném/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Modelos Animais , Mariposas/microbiologia , Mutação , Octoxinol/farmacologia , Oxacilina/farmacologia , Peptidoglicano/metabolismo , Fenótipo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Virulência , Resistência beta-Lactâmica , beta-Lactamas/farmacologia
18.
Virulence ; 12(1): 470-480, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33487122

RESUMO

We previously reported that disruption of the yjbI gene reduced virulence of Staphylococcus aureus. In this study, we found virulence in both silkworms and mice was restored by introducing the yjbH gene but not the yjbI gene to both yjbI and yjbH genes-disrupted mutants, suggesting that yjbH, the gene downstream to the yjbI gene in a two-gene operon-yjbIH, is responsible for this phenomenon. We further observed a decrease in various surface-associated proteins and changes in cell envelope glycostructures in the mutants. RNA-seq analysis revealed that disruption of the yjbI and the yjbH genes resulted in differential expression of a broad range of genes, notably, significant downregulation of genes involved in virulence and oxidative stress. Administration of N-acetyl-L-cysteine, a free-radical scavenger, restored the virulence in both the mutants. Our findings suggested that YjbH plays a role in staphylococcal pathogenicity by regulating virulence gene expression, affecting the bacterial surface structure, and conferring resistance to oxidative stress in a host.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Expressão Gênica , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Animais , Feminino , Larva/microbiologia , Camundongos , Mariposas/microbiologia , Estresse Oxidativo , Infecções Estafilocócicas/microbiologia , Virulência/genética
19.
Virulence ; 12(1): 231-243, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33410730

RESUMO

The emergence of resistance requires alternative methods to treat Candida albicans infections. We evaluated efficacy of the efflux pump inhibitor (EPI) verapamil (VER) with fluconazole (FLC) against FLC-resistant (CaR) and -susceptible C. albicans (CaS). The susceptibility of both strains to VER and FLC was determined, as well as the synergism of VER with FLC. Experiments were performed in vitro for planktonic cultures and biofilms and in vivo using Galleria mellonella. Larval survival and fungal recovery were evaluated after treatment with VER and FLC. Data were analyzed by analysis of variance and Kaplan-Meier tests. The combination of VER with FLC at sub-lethal concentrations reduced fungal growth. VER inhibited the efflux of rhodamine 123 and showed synergism with FLC against CaR. For biofilms, FLC and VER alone reduced fungal viability. The combination of VER with FLC at sub-lethal concentrations also reduced biofilm viability. In the in vivo assays, VER and FLC used alone or in combination increased the survival of larvae infected with CaR. Reduction of fungal recovery was observed only for larvae infected with CaR and treated with VER with FLC. VER reverted the FLC-resistance of C. albicans. Based on the results obtained, VER reverted the FLC-resistance of C. albicans and showed synergism with FLC against CaR. VER also increased the survival of G. mellonella infected with CaR and reduced the fungal recovery.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Fluconazol/farmacologia , Larva/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Verapamil/farmacologia , Animais , Biofilmes/efeitos dos fármacos , Transporte Biológico , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Sinergismo Farmacológico , Larva/microbiologia , Testes de Sensibilidade Microbiana , Mariposas/efeitos dos fármacos , Mariposas/microbiologia
20.
Can J Microbiol ; 67(3): 249-258, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33306436

RESUMO

Three bacterial species isolated from whole body extracts of the greater wax moth larvae, Galleria mellonella, were evaluated for their ability to utilize low-density polyethylene (LDPE) as a sole carbon source in vitro. These bacteria were identified as Lysinibacillus fusiformis, Bacillus aryabhattai, and Microbacterium oxydans. Their ability to biodegrade LDPE was assessed by growth curves, cell biomass production, polyethylene (PE) weight loss, and the presence of LDPE hydrolysis products in the growth media. Consortia of these bacteria with three other bacteria previously shown to degrade LDPE (Cupriavidus necator H16, Pseudomonas putida LS46, and Pseudomonas putida IRN22) were also tested. Growth curves of the bacteria utilizing LDPE as a sole carbon source revealed a peak in cell density after 24 h. Cell densities declined by 48 h but slowly increased again to different extents, depending on the bacteria. Incubation of LDPE with bacteria isolated from greater wax moth larvae had significant effects on bacterial cell mass production and weight loss of LDPE in PE-containing media. The bacterial consortia were better able to degrade LDPE than were the individual species alone. Gas chromatographic analyses revealed the presence of linear alkanes and other unknown putative LDPE hydrolysis products in some of bacterial culture media.


Assuntos
Bactérias/metabolismo , Consórcios Microbianos , Mariposas/microbiologia , Polietileno/metabolismo , Animais , Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Biodegradação Ambiental , Hidrólise , Larva/microbiologia
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