RESUMO
Immunoglobulin A is the dominant antibody isotype found in mucosal secretions and enforces host-microbiota symbiosis in mice, yet selective IgA-deficiency (sIgAd) in humans is often described as asymptomatic. Here, we determined the effects of IgA deficiency on human gut microbiota composition and evaluated the possibility that mucosal secretion of IgM can compensate for a lack of secretory IgA. We used 16S rRNA gene sequencing and bacterial cell sorting to evaluate gut microbiota composition and taxa-specific antibody coating of the gut microbiota in 15 sIgAd subjects and matched controls. Despite the secretion of compensatory IgM into the gut lumen, sIgAd subjects displayed an altered gut microbiota composition as compared to healthy controls. These alterations were characterized by a trend towards decreased overall microbial diversity as well as significant shifts in the relative abundances of specific microbial taxa. While secretory IgA in healthy controls targeted a defined subset of the microbiota via high-level coating, compensatory IgM in sIgAd subjects showed less specificity than IgA and bound a broader subset of the microbiota. We conclude that IgA plays a critical and non-redundant role in controlling gut microbiota composition in humans and that secretory IgA has evolved to maintain a diverse and stable gut microbial community.
Assuntos
Bactérias/classificação , Disbiose/imunologia , Imunoglobulina A Secretora/metabolismo , Imunoglobulina M/metabolismo , Adulto , Bactérias/genética , Bactérias/isolamento & purificação , Estudos de Casos e Controles , DNA Ribossômico/genética , Disbiose/microbiologia , Feminino , Humanos , Masculino , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Human gut Bacteroides use surface-exposed lipoproteins to bind and metabolize complex polysaccharides. Although vitamins and other nutrients are also essential for commensal fitness, much less is known about how commensal bacteria compete with each other or the host for these critical resources. Unlike in Escherichia coli, transport loci for vitamin B12 (cobalamin) and other corrinoids in human gut Bacteroides are replete with conserved genes encoding proteins whose functions are unknown. Here we report that one of these proteins, BtuG, is a surface-exposed lipoprotein that is essential for efficient B12 transport in B. thetaiotaomicron. BtuG binds B12 with femtomolar affinity and can remove B12 from intrinsic factor, a critical B12 transport protein in humans. Our studies suggest that Bacteroides use surface-exposed lipoproteins not only for capturing polysaccharides, but also to acquire key vitamins in the gut.
Assuntos
Bacteroides/metabolismo , Membrana Celular/metabolismo , Trato Gastrointestinal/microbiologia , Lipoproteínas/metabolismo , Vitamina B 12/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Corrinoides/metabolismo , Ligação Genética , Vida Livre de Germes , Humanos , Fator Intrínseco/metabolismo , Camundongos , Modelos Moleculares , Eletricidade EstáticaRESUMO
The secretory antibody immunoglobulin A counteracts pathogenic infections at mucosal surfaces. Recent work now reveals that IgA responses can also stabilize intestinal colonization by symbiotic microorganisms and confer resistance to future invasion by exogenous competitors.
Assuntos
Microbioma Gastrointestinal , Imunoglobulina A , Imunoglobulina A Secretora , Mucosa Intestinal , IntestinosRESUMO
In the mammalian gut, bacteria compete for resources to maintain their populations, but the factors determining their success are poorly understood. We report that the human gut bacterium Bacteroides thetaiotaomicron relies on the stringent response, an intracellular signaling pathway that allocates resources away from growth, to survive carbon starvation and persist in the gut. Genome-scale transcriptomics, 13C-labeling, and metabolomics analyses reveal that B. thetaiotaomicron uses the alarmone (p)ppGpp to repress multiple biosynthetic pathways and upregulate tricarboxylic acid (TCA) cycle genes in these conditions. During carbon starvation, (p)ppGpp triggers accumulation of the metabolite alpha-ketoglutarate, which itself acts as a metabolic regulator; alpha-ketoglutarate supplementation restores viability to a (p)ppGpp-deficient strain. These studies uncover how commensal bacteria adapt to the gut by modulating central metabolism and reveal that halting rather than accelerating growth can be a determining factor for membership in the gut microbiome.
Assuntos
Bacteroides thetaiotaomicron/fisiologia , Carbono/deficiência , Trato Gastrointestinal/microbiologia , Guanosina Pentafosfato/metabolismo , Ácidos Cetoglutáricos/metabolismo , Animais , Bacteroides thetaiotaomicron/genética , Ciclo do Ácido Cítrico/genética , Ciclo do Ácido Cítrico/fisiologia , Guanosina Pentafosfato/genética , Humanos , Metabolômica , Camundongos , Organismos Livres de Patógenos Específicos , Ácido Succínico/metabolismo , TranscriptomaRESUMO
Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. A determinant necessary for C. burnetii virulence is the Dot/Icm type IVB secretion system (T4SS). The Dot/Icm system delivers more than 100 proteins, called type IV effectors (T4Es), across the vacuolar membrane into the host cell cytosol. Several T4Es have been shown to be important for vacuolar biogenesis. Here, transposon (Tn) insertion sequencing technology (INSeq) was used to identify C. burnetii Nine Mile phase II mutants in an arrayed library, which facilitated the identification and clonal isolation of mutants deficient in 70 different T4E proteins. These effector mutants were screened in HeLa cells for deficiencies in Coxiella-containing vacuole (CCV) biogenesis. This screen identified and validated seven new T4Es that were important for vacuole biogenesis. Loss-of-function mutations in cbu0414 (coxH1), cbu0513, cbu0978 (cem3), cbu1387 (cem6), cbu1524 (caeA), cbu1752, or cbu2028 resulted in a small-vacuole phenotype. These seven mutant strains produced small CCVs in all cells tested, which included macrophage-like cells. The cbu2028::Tn mutant, though unable to develop large CCVs, had intracellular replication rates similar to the rate of the parental strain of C. burnetii, whereas the other six effector mutants defective in CCV biogenesis displayed significant reductions in intracellular replication. Vacuoles created by the cbu0513::Tn mutant did not accumulate lipidated microtubule-associated protein 1A/1B light chain 3 (LC3-II), suggesting a failure in fusion of the CCV with autophagosomes. These seven T4E proteins add to the growing repertoire of C. burnetii factors that contribute to CCV biogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Coxiella burnetii/fisiologia , Febre Q/metabolismo , Febre Q/microbiologia , Autofagossomos/metabolismo , Sistemas de Secreção Bacterianos , Coxiella burnetii/genética , Coxiella burnetii/patogenicidade , Elementos de DNA Transponíveis , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Lisossomos/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Mutação , Transporte Proteico , Vacúolos/metabolismoRESUMO
Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires' disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Legionella pneumophila/patogenicidade , Doença dos Legionários/imunologia , Animais , Proteínas de Bactérias/imunologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Legionella pneumophila/genética , Legionella pneumophila/imunologia , Doença dos Legionários/microbiologia , Mutação com Perda de Função , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Fenótipo , Virulência/genéticaRESUMO
The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes--one of two major phyla in the gut--also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.
Assuntos
Microbioma Gastrointestinal/fisiologia , Animais , Bacteroides fragilis/genética , Bacteroides fragilis/imunologia , Bacteroides fragilis/fisiologia , Feminino , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/imunologia , Genoma Bacteriano , Vida Livre de Germes , Humanos , Masculino , Camundongos , Modelos Animais , Filogenia , Simbiose/genética , Simbiose/imunologia , Simbiose/fisiologia , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/imunologia , Sistemas de Secreção Tipo VI/fisiologiaRESUMO
What regulates chromosome segregation dynamics in bacteria is largely unknown. Here, we show in Caulobacter crescentus that the polarity factor TipN regulates the directional motion and overall translocation speed of the parS/ParB partition complex by interacting with ParA at the new pole. In the absence of TipN, ParA structures can regenerate behind the partition complex, leading to stalls and back-and-forth motions of parS/ParB, reminiscent of plasmid behaviour. This extrinsic regulation of the parS/ParB/ParA system directly affects not only division site selection, but also cell growth. Other mechanisms, including the pole-organizing protein PopZ, compensate for the defect in segregation regulation in ΔtipN cells. Accordingly, synthetic lethality of PopZ and TipN is caused by severe chromosome segregation and cell division defects. Our data suggest a mechanistic framework for adapting a self-organizing oscillator to create motion suitable for chromosome segregation.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Ciclo Celular/fisiologia , Polaridade Celular , Segregação de Cromossomos , Cromossomos Bacterianos/fisiologia , Proteínas de Bactérias/genética , Western Blotting , Caulobacter crescentus/fisiologia , Imunoprecipitação , Origem de ReplicaçãoRESUMO
Polarity is often an intrinsic property of the cell, yet little is known about its origin or its maintenance over generations. Here we identify a landmark protein, TipN, which acts as a spatial and temporal cue for setting up the correct polarity in the bacterium Caulobacter crescentus. TipN marks the new pole throughout most of the cell cycle, and its relocation to the nascent poles at the end of division provides a preexisting reference point for orienting the polarity axis in the progeny. Deletion of tipN causes pleiotropic polarity defects, including frequently reversed asymmetry in progeny size and mislocalization of proteins and organelles. Ectopic localization of TipN along the lateral side of the cell creates new axes of polarity leading to cell branching and formation of competent cell poles. Localization defects of the actin-like protein MreB in the DeltatipN mutant suggest that TipN is upstream of MreB in regulating cell polarity.