RESUMO
Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are trafficked from the bacterium to the host via unknown mechanisms. Arabinomannan is thought to be a capsular derivative of these molecules, lacking a lipid anchor. However, the mechanism by which this material is generated has yet to be elucidated. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH (Rv0365c in Mycobacterium tuberculosis) which specifically cleaves α-1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures, potentially implicating arabinomannan as a signal for growth phase transition. Finally, we demonstrate that LamH is important for M. tuberculosis survival in macrophages.
Assuntos
Proteínas de Bactérias , Glicosídeo Hidrolases , Lipopolissacarídeos , Macrófagos , Mananas , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Lipopolissacarídeos/metabolismo , Mananas/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Glicosídeo Hidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Animais , Camundongos , Humanos , Fosfatidilinositóis/metabolismo , Cápsulas Bacterianas/metabolismoRESUMO
Capsular polysaccharide (CPS) as a probiotic component has the ability to regulate the function of the host's immune system. However, how the structure and function of heat-killed CPS are altered remains unclear. In the present study, CPS were isolated and purified from live (LCPS) and heat-killed (HCPS) Lacticaseibacillus paracasei 6235. The differences in structure and immunomodulation between LCPS and HCPS were compared and analyzed. The results demonstrate that after heat killed, the molecular weight of CPS decreased from 23.4 kDa to 17.5 kDa, with the disappearance of galactosamine in the monosaccharide composition, and changes in the microstructure. Methylation analysis and nuclear magnetic resonance analysis revealed that the LCPS and HCPS are similar in structure, which main units of â3,4)-α-D-Glcp-(1â4)-α-D-Galp-(1â3)-ß-L-Rhap-(1â6)-ß-D-Galp-(1â, and repeating units of â3,4)-α-D-Glcp-(1â, â3)-ß-L-Rhap-(1â, and â4)-α-D-Galp-(1â residues. Furthermore, both LCPS and HCPS significantly downregulated the expression of pro-inflammatory cytokines in RAW264.7 cells induced by LPS. Specifically, HCPS reduced the levels of IL-6 and IL-1ß by 79.38 % and 88.42 %, respectively, compared to LCPS. Concurrently, both LCPS and HCPS effectively mitigated inflammatory responses through the NF-κB and MAPK signaling pathways. Moreover, compared to LCPS, HCPS increased the protein expression levels of NF-κB/p-NF-κB and IκB/p-IκB by 26.14 % and 28.92 %, respectively. These results suggest that CPS has a role in modulating immune responses and that HCPS is more effective. This study can be further developed into new products related to postbiotics.
Assuntos
Temperatura Alta , Lacticaseibacillus paracasei , Polissacarídeos Bacterianos , Camundongos , Animais , Células RAW 264.7 , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/farmacologia , Lacticaseibacillus paracasei/química , Peso Molecular , NF-kappa B/metabolismo , Cápsulas Bacterianas/química , Cápsulas Bacterianas/imunologia , Interleucina-6/metabolismo , Citocinas/metabolismo , MetilaçãoRESUMO
The hyaluronic acid capsule is crucial in protecting group A Streptococcus (GAS) against phagocytic killing. However, there have been reported outbreaks caused by capsule-deficient GAS strains, and the mechanisms underlying their evasion of immune clearance remain unclear. This study demonstrated that the capsule-deficient mutant [Cap(-)] of the emm1 strain increased survival within phagocytic cells compared to the wild-type strain [Cap(+)]. Although both Cap(+) and Cap(-) strains exhibited similar abilities to disrupt the phagosome, only the Cap(+) strain was colocalized with lysosomes and acidified compartments in phagocytic cells, indicating its susceptibility to autophagosome elimination. In contrast, the Cap(-) mutant evaded the recognition of galectin-8 and ubiquitin, impairing selective autophagy-mediated elimination. These findings suggest that a deficiency in the capsule could impair the intracellular elimination of GAS in macrophages, revealing previously unknown aspects of the host's recognition of the GAS capsule in macrophages. IMPORTANCE: Group A Streptococcus (GAS) is a Gram-positive bacterium that causes diseases ranging from mild pharyngitis to severe necrotizing fasciitis. Phagocytic cells serve as the primary defense against bacterial infections, exhibiting remarkable efficiency in eliminating intracellular pathogens. The hyaluronic acid capsule is a critical virulence factor that contributes to the resistance of phagocytosis in GAS. Nevertheless, the outbreaks caused by GAS strains that lack the hyaluronic acid capsule have been reported, and the selective advantage of capsule-deficient strains during infection is not fully understood. This study showed that the autophagic adaptor proteins recognize the capsulated GAS strain but not the capsule-deficient mutant, indicating that the hyaluronic acid capsule could be the autophagic target in macrophages. These findings imply that the hyaluronic acid capsule of GAS actually enhances its elimination within phagocytic cells, subverting the understanding of the capsule in GAS pathogenesis.
Assuntos
Autofagia , Cápsulas Bacterianas , Macrófagos , Streptococcus pyogenes , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/fisiologia , Macrófagos/microbiologia , Macrófagos/imunologia , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/genética , Humanos , Evasão da Resposta Imune , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/imunologia , Fagocitose , Camundongos , Ácido Hialurônico/metabolismo , AnimaisRESUMO
During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1ß, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1ß, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.
Assuntos
Citocinas , Células Dendríticas , Porphyromonas gingivalis , Células Th17 , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Porphyromonas gingivalis/imunologia , Humanos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Células Th17/imunologia , Células Th17/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Citocinas/metabolismo , Diferenciação Celular , Células Th1/imunologia , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Receptor Notch2/genética , Receptor Notch2/metabolismo , Células Cultivadas , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/metabolismo , Infecções por Bacteroidaceae/imunologia , Infecções por Bacteroidaceae/microbiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.
Assuntos
Cápsulas Bacterianas , Proteínas de Escherichia coli , Escherichia coli , Polissacarídeos Bacterianos , Trifosfato de Adenosina/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/química , Cápsulas Bacterianas/ultraestrutura , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/química , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Glicolipídeos/química , Glicolipídeos/metabolismo , Hidrólise , Microscopia de Fluorescência , Modelos Moleculares , Polissacarídeos Bacterianos/metabolismo , Polissacarídeos Bacterianos/química , Especificidade por SubstratoRESUMO
Klebsiella pneumoniae produces a thick capsule layer composed of extracellular polysaccharides protecting the bacterial cells from clearance by innate host immunity during infection. Here we characterize the interactions of a structurally diverse set of host defense peptides with K. pneumoniae extracellular polysaccharides. Remarkably, we found that all host defense peptides were active against a diverse set of K. pneumoniae strains, including hypermucoviscous strains with extensive capsule production, and aggregated with extracted capsule. Interestingly, the polyproline peptide bac7 (1-35), was the most potent antimicrobial and induced the most capsule aggregation. In addition to capsule aggregation, we found that bac7 (1-35) could also disrupt pre-formed hypermucoviscous K. pneumoniae biofilm. Further analysis using scanning electron microscopy revealed the biofilm matrix of a hypermucoviscous strain is removed by bac7 (1-35) exposing associated bacterial cells. This is the first description of a host defense peptide interacting with capsular and biofilm extracellular polysaccharides to expose cells from a K. pneumoniae biofilm matrix and suggests that features of polyproline peptides may be uniquely suited for extracellular polysaccharide interactions. IMPORTANCE Klebsiella pneumoniae bacterial infections are a major threat to human health as mortality rates are steadily on the rise. A defining characteristic of K. pneumoniae is the robust polysaccharide capsule that aids in resistance to the human immune system. We have previously discovered that a synthetic peptide could aggregate with capsule polysaccharides and disrupt the capsule of K. pneumoniae. Here we describe that host defense peptides also aggregate with capsule produced from hypermucoviscous K. pneumoniae, revealing this mechanism is shared by natural peptides. We found the polyproline peptide bac7 (1-35) had the greatest antimicrobial activity and caused the most capsule aggregation. Interestingly, bac7 (1-35) also removed the biofilm matrix of hypermucoviscous K. pneumoniae exposing the associated bacterial cells. This is the first description of a polyproline peptide interacting with capsular and biofilm polysaccharides to expose cells from a K. pneumoniae biofilm matrix.
Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Peptídeos Catiônicos Antimicrobianos , Cápsulas Bacterianas , Biofilmes , Humanos , Infecções por Klebsiella/microbiologia , Peptídeos , Polissacarídeos , Polissacarídeos BacterianosRESUMO
Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019, and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes into close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic, innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional, with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay shows that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows a lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status, determined by factors such as age, might play an important role in determining infection outcome.
Assuntos
Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Adolescente , Adulto , Fatores Etários , Idoso , Líquido da Lavagem Broncoalveolar , Estruturas Celulares , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/genética , Masculino , Manosídeos/biossíntese , Manosídeos/genética , Manosiltransferases/biossíntese , Manosiltransferases/genética , Pessoa de Meia-Idade , Adulto JovemRESUMO
Campylobacter jejuni is a Gram-negative, pathogenic bacterium that is commensal in poultry. Infection of C. jejuni leads to campylobacteriosis, the leading cause of gastroenteritis worldwide. Coating the surface of C. jejuni is a thick layer of sugar molecules known as the capsular polysaccharide (CPS). The CPS of C. jejuni NCTC 11168 (HS:2) is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronate, d-N-acetyl-galactosamine, and d-ribose. The glucuronate is further amidated with either ethanolamine or serinol, but it is unknown how this new amide bond is formed. Sequence similarity networks were used to identify a candidate enzyme for amide bond formation during the biosynthesis of the CPS of C. jejuni. The C-terminal domain of Cj1438 was shown to catalyze amide bond formation using MgATP and d-glucuronate in the presence of either ethanolamine phosphate or (S)-serinol phosphate. Product formation was verified using 31P NMR spectroscopy and ESI mass spectrometry, and the kinetic constants determined using a coupled enzyme assay by measuring the rate of ADP formation. This work represents the first functional characterization of an ATP-dependent amidoligase in the formation of amide bonds in the biosynthetic pathway for the assembly of the CPS in C. jejuni.
Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/metabolismo , Polissacarídeos Bacterianos/metabolismo , Trifosfato de Adenosina/metabolismo , Vias Biossintéticas , Infecções por Campylobacter/microbiologia , HumanosRESUMO
Escherichia coli K1 causes bacteremia and meningitis in human neonates. The K1 capsule, an α2,8-linked polysialic acid (PSA) homopolymer, is its essential virulence factor. PSA is usually partially modified by O-acetyl groups. It is known that O-acetylation alters the antigenicity of PSA, but its impact on the interactions between E. coli K1 and host cells is unclear. In this study, a phase variant was obtained by passage of E. coli K1 parent strain, which expressed a capsule with 44% O-acetylation whereas the capsule of the parent strain has only 3%. The variant strain showed significantly reduced adherence and invasion to macrophage-like cells in comparison to the parent strain. Furthermore, we found that O-acetylation of PSA enhanced the modulation of trafficking of E. coli-containing vacuoles (ECV), enabling them to avoid fusing with lysosomes in these cells. Intriguingly, by using quartz crystal microbalance, we demonstrated that the PSA purified from the parent strain interacted with human sialic acid-binding immunoglobulin-like lectins (Siglecs), including Siglec-5, Siglec-7, Siglec-11, and Siglec-14. However, O-acetylated PSA from the variant interacted much less and also suppressed the production of Siglec-mediated proinflammatory cytokines. The adherence of the parent strain to human macrophage-like cells was significantly blocked by monoclonal antibodies against Siglec-11 and Siglec-14. Furthermore, the variant strain caused increased bacteremia and higher lethality in neonatal mice compared to the parent strain. These data elucidate that O-acetylation of K1 capsule enables E. coli to escape from Siglec-mediated innate immunity and lysosomal degradation; therefore, it is a strategy used by E. coli K1 to regulate its virulence. IMPORTANCE Escherichia coli K1 is a leading cause of neonatal meningitis. The mortality and morbidity of this disease remain significantly high despite antibiotic therapy. One major limitation on advances in prevention and therapy for meningitis is an incomplete understanding of its pathogenesis. E. coli K1 is surrounded by PSA, which is observed to have high-frequency variation of O-acetyl modification. Here, we present an in-depth study of the function of O-acetylation in PSA at each stage of host-pathogen interaction. We found that a high level of O-acetylation significantly interfered with Siglec-mediated bacterial adherence to macrophage-like cells, and blunted the proinflammatory response. Furthermore, the O-acetylation of PSA modulated the trafficking of ECVs to prevent them from fusing with lysosomes, enabling them to escape degradation by lysozymes within these cells. Elucidating how subtle modification of the capsule enhances bacterial defenses against host innate immunity will enable the future development of effective drugs or vaccines against infection by E. coli K1.
Assuntos
Cápsulas Bacterianas/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Ácidos Siálicos/imunologia , Acetilação , Animais , Escherichia coli/genética , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Imunidade Inata , Lisossomos/imunologia , Lisossomos/microbiologia , Masculino , Camundongos , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Vacúolos/imunologia , Vacúolos/microbiologiaRESUMO
The capsular polysaccharide (CPS) of Streptococcus suis defines various serotypes based on its composition and structure. Though serotype switching has been suggested to occur between S. suis strains, its impact on pathogenicity and virulence remains unknown. Herein, we experimentally generated S. suis serotype-switched mutants from a serotype 2 strain that express the serotype 3, 4, 7, 8, 9, or 14 CPS. The effects of serotype switching were then investigated with regards to classical properties conferred by presence of the serotype 2 CPS, including adhesion to/invasion of epithelial cells, resistance to phagocytosis by macrophages, killing by whole blood, dendritic cell-derived pro-inflammatory mediator production and virulence using mouse and porcine infection models. Results demonstrated that these properties on host cell interactions were differentially modulated depending on the switched serotypes, although some different mutations other than loci of CPS-related genes were found in each the serotype-switched mutant. Among the serotype-switched mutants, the mutant expressing the serotype 8 CPS was hyper-virulent, whereas mutants expressing the serotype 3 or 4 CPSs had reduced virulence. By contrast, switching to serotype 7, 9, or 14 CPSs had little to no effect. These findings suggest that serotype switching can drastically alter S. suis virulence and host cell interactions.
Assuntos
Cápsulas Bacterianas/imunologia , Interações Hospedeiro-Patógeno , Sorogrupo , Streptococcus suis/genética , Animais , Cápsulas Bacterianas/genética , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Streptococcus suis/imunologia , Streptococcus suis/patogenicidade , Virulência/genéticaRESUMO
The capsule of Bacillus anthracis is composed of a d isomer poly-γ-glutamic acid polymer, which is especially nonstimulatory to dendritic cells, even more so than similar mixed d, l isomer polymers from nonpathogenic Bacillus species. Capsule is an essential virulence factor for B. anthracis, protecting the bacilli from phagocytosis by innate immune cells. In this study, we demonstrate that encapsulation provides a further pathogenic advantage by shielding more inflammatory Ags on the bacillus surface, thereby reducing dendritic cell responses. We exposed human immature dendritic cells (DCs) to increasing multiplicities of infection (MOIs) of killed B. anthracis bacilli from the fully encapsulated wild-type Ames strain (WT) and an isogenic capsule-deficient strain (capA mutant). Both strains elicited robust cytokine responses, but IL-23, TNF-α, and IL-10 were significantly reduced in response to the encapsulated WT compared with capA mutant up to an MOI of 15. capA mutant bacilli could induce phenotypic maturation of immature DCs with upregulation of MHC classes I and II, CD83, and CCR7 at an MOI of 3.75, whereas encapsulated WT bacilli still did not induce significant upregulation of MHC classes I and II at an MOI of 15. DCs exposed to capA mutant bacilli (MOI 3.75) exhibited CCR7-dependent chemotaxis that was comparable to that of LPS-stimulated controls, whereas DCs exposed to encapsulated WT bacilli exhibited significantly less chemotaxis. We conclude that capsule shields more inflammatory surface Ags, delaying development of an adaptive immune response by reducing TNF-α, thereby inhibiting DC maturation.
Assuntos
Bacillus anthracis/imunologia , Cápsulas Bacterianas/imunologia , Células Dendríticas/imunologia , Macrófagos/imunologia , Ácido Poliglutâmico/análogos & derivados , Citocinas/metabolismo , Humanos , Imunidade Inata , Fagocitose , Ácido Poliglutâmico/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. RESULTS: Analysis of intra- and extracellular changes identified 2380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. CONCLUSIONS: This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.
Assuntos
Cápsulas Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Proteômica , Transcrição Gênica , Zinco/metabolismo , Animais , Cápsulas Bacterianas/fisiologia , Proteínas de Bactérias/genética , Klebsiella pneumoniae/química , Klebsiella pneumoniae/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Proteoma , Virulência/genética , Fatores de Virulência/genética , Zinco/farmacologiaRESUMO
Group B Streptococcus (GBS) is an opportunistic bacterial pathogen that can contribute to the induction of preterm birth in colonized pregnant women and to severe neonatal disease. Many questions regarding the mechanisms that drive GBS-associated pathogenesis remain unanswered, and it is not yet clear why virulence has been observed to vary so extensively across GBS strains. Previously, we demonstrated that GBS strains of different sequence types (STs) and capsule (CPS) types induce different cytokine profiles in infected THP-1 macrophage-like cells. Here, we expanded on these studies by utilizing the same set of genetically diverse GBS isolates to assess ST and CPS-specific differences in upstream cell death and inflammatory signaling pathways. Our results demonstrate that particularly virulent STs and CPS types, such as the ST-17 and CPS III groups, induce enhanced Jun-N-terminal protein kinase (JNK) and NF-κB pathway activation following GBS infection of macrophages compared with other ST or CPS groups. Additionally, we found that ST-17, CPS III, and CPS V GBS strains induce the greatest levels of macrophage cell death during infection and exhibit a more pronounced ability to be internalized and to survive in macrophages following phagocytosis. These data provide further support for the hypothesis that variable host innate immune responses to GBS, which significantly impact pathogenesis, stem in part from genotypic and phenotypic differences among GBS isolates. These and similar studies may inform the development of improved diagnostic, preventive, or therapeutic strategies targeting invasive GBS infections.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Macrófagos/imunologia , Transdução de Sinais , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/fisiologia , Estresse Fisiológico , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/metabolismo , Biomarcadores , Suscetibilidade a Doenças , Humanos , Imunidade Inata , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/metabolismo , Células THP-1RESUMO
Antibacterial resistance (AR) is causing more and more bacterial infections that cannot be cured by using the antibacterial drugs that are currently available. It is predicted that 10 million people will die every year by 2050 from infections caused by antibacterial resistant strains, surpassing the predicted numbers of deaths caused by cancer. AR is therefore a global challenge and novel antibacterial strategies are in high demand. To this end, the work on exploring the pore properties of a bacterial sugar transporter, WzaK30, has led to the discovery of the first inhibitor against bacterial capsular polysaccharides export.Recently, single-molecule recapitulation of capsular polysaccharide (CPS) export and pore formation properties of Wza barrel peptides have also revealed the possibility of a next-generation of Wza strategies. These strategies are based upon the first examination and understanding of the pore properties of wild-type (WT) and mutant WzaK30 in single-molecule electrical channel recording. The initially reported experimental procedures have been further developed to enable efficient studies of other Wza homologs that are more common in bacterial pathogens causing significant bacterial infections. Therefore, this chapter presents the most recent protocols and logistics behind the research on Wza channel activity, antibacterials, and strategies. The disciplines covered here include computation, molecular biology, biochemistry, electrophysiology, microbiology, and biophysics.
Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Polissacarídeos Bacterianos/metabolismo , Antibacterianos/farmacologia , Cápsulas Bacterianas/efeitos dos fármacos , Transporte Biológico , Escherichia coli/efeitos dos fármacosRESUMO
STREPTOCOCCUS SUIS: serotype 2 (S. suis 2) is an important swine pathogen and also an emerging zoonotic agent. HtpsA has been reported as an immunogenic cell surface protein on the bacterium. In the present study, we constructed an isogenic mutant strain of htpsA, namely ΔhtpsA, to study its role in the development and virulence of S. suis 2. Our results showed that the mutant strain lost its typical encapsulated structure with decreased concentrations of sialic acid. Furthermore, the survival rate in whole blood, the anti-phagocytosis by RAW264.7 murine macrophage, and the adherence ability to HEp-2 cells were all significantly affected in the ΔhtpsA. In addition, the deletion of htpsA sharply attenuated the virulence of S. suis 2 in an infection model of mouse. RNA-seq analysis revealed that 126 genes were differentially expressed between the ΔhtpsA and the wild-type strains, including 28 upregulated and 98 downregulated genes. Among the downregulated genes, many were involved in carbohydrate metabolism and synthesis of virulence-associated factors. Taken together, htpsA was demonstrated to play a role in the morphological development and pathogenesis of the highly virulent S. suis 2 05ZYH33 strain.
Assuntos
Cápsulas Bacterianas/fisiologia , Proteínas de Bactérias/genética , Inativação Gênica , Streptococcus suis/genética , Streptococcus suis/patogenicidade , Fatores de Virulência/genética , Animais , Aderência Bacteriana/genética , Feminino , Humanos , Macrófagos/microbiologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/genética , Mutação , Fagocitose , Células RAW 264.7 , Sorogrupo , Organismos Livres de Patógenos Específicos , Infecções Estreptocócicas/microbiologia , Streptococcus suis/classificação , Virulência/genéticaRESUMO
Biofilm formation by Streptococcus pyogenes (group A streptococcus [GAS]) in model systems mimicking the respiratory tract is poorly documented. Most studies have been conducted on abiotic surfaces, which poorly represent human tissues. We have previously shown that GAS forms mature and antibiotic-resistant biofilms on physiologically relevant epithelial cells. However, the roles of the substratum, extracellular matrix (ECM) components, and GAS virulence factors in biofilm formation and structure are unclear. In this study, biofilm formation was measured on respiratory epithelial cells and keratinocytes by determining biomass and antibiotic resistance, and biofilm morphology was visualized using scanning electron microscopy. All GAS isolates tested formed biofilms that had similar, albeit not identical, biomass and antibiotic resistance for both cell types. Interestingly, functionally mature biofilms formed more rapidly on keratinocytes but were structurally denser and coated with more ECM on respiratory epithelial cells. The ECM was crucial for biofilm integrity, as protein- and DNA-degrading enzymes induced bacterial release from biofilms. Abiotic surfaces supported biofilm formation, but these biofilms were structurally less dense and organized. No major role for M protein, capsule, or streptolysin O was observed in biofilm formation on epithelial cells, although some morphological differences were detected. NAD-glycohydrolase was required for optimal biofilm formation, whereas streptolysin S and cysteine protease SpeB impaired this process. Finally, no correlation was found between cell adherence or autoaggregation and GAS biofilm formation. Combined, these results provide a better understanding of the role of biofilm formation in GAS pathogenesis and can potentially provide novel targets for future treatments against GAS infections.
Assuntos
Biofilmes/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Antibacterianos/farmacologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Biofilmes/efeitos dos fármacos , Biomassa , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Farmacorresistência Bacteriana , Células Epiteliais/ultraestrutura , Matriz Extracelular/microbiologia , Matriz Extracelular/ultraestrutura , Queratinócitos/microbiologia , Queratinócitos/ultraestrutura , Sorogrupo , Fatores de Virulência/genéticaRESUMO
Iron is essential for the growth of the human fungal pathogen Cryptococcus neoformans within the vertebrate host, and iron sensing contributes to the elaboration of key virulence factors, including the formation of the polysaccharide capsule. C. neoformans employs sophisticated iron acquisition and utilization systems governed by the transcription factors Cir1 and HapX. However, the details of the transcriptional regulatory networks that are governed by these transcription factors and connections to virulence remain to be defined. Here, we used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and transcriptome analysis (RNA-seq) to identify genes directly regulated by Cir1 and/or HapX in response to iron availability. Overall, 40 and 100 genes were directly regulated by Cir1, and 171 and 12 genes were directly regulated by HapX, under iron-limited and replete conditions, respectively. More specifically, we found that Cir1 directly controls the expression of genes required for iron acquisition and metabolism, and indirectly governs capsule formation by regulating specific protein kinases, a regulatory connection not previously revealed. HapX regulates the genes responsible for iron-dependent pathways, particularly under iron-depleted conditions. By analyzing target genes directly bound by Cir1 and HapX, we predicted the binding motifs for the transcription factors and verified that the purified proteins bind these motifs in vitro Furthermore, several direct target genes were coordinately and reciprocally regulated by Cir1 and HapX, suggesting that these transcription factors play conserved roles in the response to iron availability. In addition, biochemical analyses revealed that Cir1 and HapX are iron-containing proteins, implying that the regulatory networks of Cir1 and HapX may be influenced by the incorporation of iron into these proteins. Taken together, our identification of the genome-wide transcriptional networks provides a detailed understanding of the iron-related regulatory landscape, establishes a new connection between Cir1 and kinases that regulate capsule, and underpins genetic and biochemical analyses that reveal iron-sensing mechanisms for Cir1 and HapX in C. neoformans.
Assuntos
Cápsulas Bacterianas/fisiologia , Criptococose/metabolismo , Cryptococcus neoformans/fisiologia , Proteínas Fúngicas/metabolismo , Homeostase , Ferro/fisiologia , Transcrição Gênica , Criptococose/genética , Criptococose/microbiologia , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Humanos , VirulênciaRESUMO
Bacillus anthracis poly-γ-D-glutamic acid (PGA) capsule is an essential virulent factor that helps the bacterial pathogen to escape host immunity. Like other encapsulated bacterial species, the B. anthracis capsule may also inhibit complement-mediated clearance and ensure bacterial survival in the host. Previous reports suggest that B. anthracis spore proteins inhibit complement activation. However, the mechanism through which the B. anthracis capsule imparts a survival advantage to the active bacteria has not been demonstrated till date. Thus, to evaluate the role of the PGA capsule in evading host immunity, we have undertaken the present head-to-head comparative study of the phagocytosis and complement activation of non-encapsulated and encapsulated B. anthracis strains. The encapsulated virulent strain exhibited resistance toward complement-dependent and complement-independent bacterial phagocytosis by human macrophages. The non-encapsulated Sterne strain was highly susceptible to phagocytosis by THP-1 macrophages, after incubation with normal human serum (NHS), heat-inactivated serum, and serum-free media, thus indicating that the capsule inhibited both complement-dependent and complement-independent opsonic phagocytosis. An increased binding of C3b and its subsequent activation to C3c and C3dg, which functionally act as potent opsonins, were observed with the non-encapsulated Sterne strain compared with the encapsulated strain. Other known mediators of complement fixation, IgG, C-reactive protein (CRP), and serum amyloid P component (SAP), also bound more prominently with the non-encapsulated Sterne strain. Studies with complement pathway-specific, component-deficient serum demonstrated that the classical pathway was primarily involved in mediating C3b binding on the non-encapsulated bacteria. Both strains equally bound the complement regulatory proteins C4BP and factor H. Importantly, we demonstrated that the negative charge of the PGA capsule was responsible for the differential binding of the complement proteins between the non-encapsulated and encapsulated strains. At lower pH closer to the isoelectric point of PGA, the neutralization of the negative charge was associated with an increased binding of C3b and IgG with the encapsulated B. anthracis strain. Overall, our data have demonstrated that the B. anthracis capsule inhibits complement fixation and opsonization resulting in reduced phagocytosis by macrophages, thus allowing the bacterial pathogen to evade host immunity.
Assuntos
Antraz/imunologia , Bacillus anthracis/fisiologia , Macrófagos/imunologia , Ácido Poliglutâmico/análogos & derivados , Antígenos de Bactérias/imunologia , Bacillus anthracis/patogenicidade , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/metabolismo , Ativação do Complemento , Complemento C3b/metabolismo , Humanos , Evasão da Resposta Imune , Proteínas Opsonizantes/metabolismo , Fagocitose , Ácido Poliglutâmico/metabolismo , Ligação Proteica , Células THP-1 , VirulênciaRESUMO
Pasteurella multocida is a gram-negative bacterial pathogen, which causes a large number of diseases in mammals, birds and human. Although the bacterium has been known for decades, the pathogenesis and the mechanisms of P. multocida induced host immunity are poorly understood. Recently, we have reported that nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome plays an important role in caspase-1 activation and IL-1ß secretion in macrophages infected with P. multocida. In this study, the inflammasome activation and IL-1ß secretion were further demonstrated by using high- and low-virulent bovine P. multocida isolates. The results showed that, comparing with macrophages infected with the high-virulent PmCQ2 isolates, the low-virulent PmCQ6 induced higher levels of NLRP3 transcription, caspase-1 activation and mature IL-1ß secretion. Furthermore, the capsule of the high-virulent PmCQ2 was much thicker than that of low-virulent PmCQ6, which indicating that capsular thickness might influence the bacteria colonization and NLRP3 inflammasome activation. The results suggested that differences in maturation of IL-1ß in macrophages upon high- and low- virulent P. multocida infection are critically dependent on the differential activation of NLRP3 inflammasome. This study provided more understanding for the host immune responses induced by P. multocida and further extended the knowledge of P. multocida virulence from the view of host innate immunity.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Inflamassomos/imunologia , Interleucina-1beta/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Pasteurella multocida/patogenicidade , Animais , Cápsulas Bacterianas/imunologia , Caspase 1/imunologia , Imunidade Inata , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Organismos Livres de Patógenos Específicos , VirulênciaRESUMO
Cryptococcus neoformans, an encapsulated fungal pathogen is evolving as a major threat to immune-compromised patients and rarely to healthy individuals also. The cell wall bound capsular polysaccharide, melanin pigment and biofilm formation are major virulence factors that are known to contribute to cryptococcal meningitis. In the present study, a furanone derivative, (E)-5-benzylidenedihydrofuran-2(3H)-one (compound-6) was evaluated against biofilm of seven different strains of C. neoformans in melanized and non-melanized condition. In addition, the efficacy of compound-6 in activation of TLR-2, opsonophagocytosis, and modulation of cytokine expression during phagocytosis were studied. During the biofilm study, we found that moderate capsule size favored biofilm formation. Interestingly, the minimum biofilm eradication concentration (MBEC0.5) of melanized biofilm was found to be achieved at 1- to 1.7-fold higher MBEC0.5 of non-melanized cells. The maximum eradication of 77% and 69% of non-melanized and melanized biofilm were observed. The capsule size was reduced to half of its size with marked changes in morphology. Furthermore, expression of TLR2, iNOS and pro-inflammatory cytokines such as TNF-α, IL-12, and IFN-γ were also facilitated by compound-6. The correlation analysis showed a positive correlation between phagocytosis and the expression of TLR-2, iNOS, IL-6, IL-12. Collectively, the significant effect of compound-6, anti-melanization activity, antibiofilmand effective immunomodulant could be an interesting dual strategy drug agonist against cryptococcal meningitis.