RESUMO
The heterogeneity of bacterial growth and replicative rates within a population was proposed a century ago notably to explain the presence of bacterial persisters. The term "growth rate" at the single-cell level corresponds to the increase in size or mass of an individual bacterium while the "replicative rate" refers to its division capacity within a defined temporality. After a decades long hiatus, recent technical innovative approaches allow population growth and replicative rates heterogeneity monitoring at the single-cell level resuming in earnest. Among these techniques, the oldest and widely used is time-lapse microscopy, most recently combined with microfluidics. We also discuss recent fluorescence dilution methods informing only on replicative rates and best suited. Some new elegant single cell methods so far only sporadically used such as buoyant mass measurement and stable isotope probing have emerged. Overall, such tools are widely used to investigate and compare the growth and replicative rates of bacteria displaying drug-persistent behaviors to that of bacteria growing in specific ecological niches or collected from patients. In this review, we describe the current methods available, discussing both the type of queries these have been used to answer and the specific strengths and limitations of each method.
Assuntos
Microfluídica , Microscopia , Humanos , Microfluídica/métodos , Replicação do DNA , BactériasRESUMO
Staphylococcus aureus - a major aetiological agent of bone and joint infection (BJI) - is associated with a high risk of relapse and chronicity, in part due to its ability to invade and persist in non-professional phagocytic bone cells such as osteoblasts. This intracellular reservoir protects S. aureus from the action of the immune system and most antibiotics. To date, the choice of antimicrobial strategies for BJI treatment mostly relies on standard susceptibility testing, bone penetration of antibiotics and their 'antibiofilm' activity. Despite the role of intracellular persistent S. aureus in the development of chronic infection, the ability of antibiotics to target the S. aureus intraosteoblastic reservoir is not considered in therapeutic choices but might represent a key determinant of treatment outcome. This review provides an overview of the intracellular pharmacokinetics of antistaphylococcal drugs used in the treatment of BJI and of their ability to target intraosteoblastic S. aureus. Thirteen studies focusing on the intraosteoblastic activity of antibiotics against S. aureus were reviewed, all relying on in vitro models of osteoblast infection. Despite varying incubation times, multiplicities of infection, bacterial strains, and the types of infected cell lines, rifamycins and fluoroquinolones remain the two most potent antimicrobial classes for intraosteoblastic S. aureus eradication, consistent with clinical data showing a superiority of this combination therapy in S. aureus orthopaedic device-related infections.
Assuntos
Rifamicinas , Infecções Estafilocócicas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Infecção Persistente , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureusRESUMO
As of 23 April 2021, the outbreak of COVID-19 claimed around 150 million confirmed cases with over 3 million deaths worldwide. Yet, an even more serious but silent pandemic, that of antimicrobial resistance (AMR), is likely complicating the outcome of COVID-19 patients. This study discusses the current knowledge on the emergence of the SARS-CoV-2 and highlights the likely contribution of the COVID-19 pandemic on the escalation of AMR. COVID-19 engenders extensive antibiotic overuse and misuse, and will undoubtedly and substantially increase AMR rates worldwide. Amid the expanding COVID-19 pandemic, policymakers should consider the hidden threat of AMR much more, which may well be enhanced through improper use of antibiotics to treat patients with severe COVID-19 infection.
RESUMO
In Streptococcus pneumoniae TIGR4, genes encoding a SecY2A2 accessory Sec system are present within a locus encoding a serine-rich repeat surface protein PsrP. Mutant strains deleted in secA2 or psrP were deficient in biofilm formation, while the ΔsecA2 mutant was reduced in binding to airway epithelial cells. Cell wall protein (CWP) fractions from the ΔsecA2 mutant, but not from the ΔpsrP mutant, were reduced in haemolytic (pneumolysin) activity. Contact-dependent pneumolysin (Ply) activity of wild type TIGR4 cells was ten-fold greater than that of ΔsecA2 mutant cells suggesting that Ply was not active at the ΔsecA2 cell surface. Ply protein was found to be present in the CWP fraction from the ΔsecA2 mutant, but showed aberrant electrophoretic migration indicative of protein modification. Proteomic analyses led to the discovery that the ΔsecA2 mutant CWP fraction was deficient in two glycosidases as well as other enzymes involved in carbohydrate metabolism. Taken collectively the results suggest that positioning of Ply into the cell wall compartment in active form, together with glycosyl hydrolases and adhesins, requires a functional accessory Sec system.
Assuntos
Aderência Bacteriana , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/metabolismo , Biofilmes/crescimento & desenvolvimento , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Estreptolisinas/metabolismo , Proteínas de Bactérias/metabolismo , Deleção de Genes , Humanos , Streptococcus pneumoniae/fisiologiaRESUMO
Shigella is the major cause of bacillary dysentery world-wide. It is divided into four species, named S. flexneri, S. sonnei, S. dysenteriae, and S. boydii, which are distinct genomically and in their ability to cause disease. Shigellosis, the clinical presentation of Shigella infection, is characterized by watery diarrhea, abdominal cramps, and fever. Shigella's ability to cause disease has been attributed to virulence factors, which are encoded on chromosomal pathogenicity islands and the virulence plasmid. However, information on these virulence factors is not often brought together to create a detailed picture of infection, and how this translates into shigellosis symptoms. Firstly, Shigella secretes virulence factors that induce severe inflammation and mediate enterotoxic effects on the colon, producing the classic watery diarrhea seen early in infection. Secondly, Shigella injects virulence effectors into epithelial cells via its Type III Secretion System to subvert the host cell structure and function. This allows invasion of epithelial cells, establishing a replicative niche, and causes erratic destruction of the colonic epithelium. Thirdly, Shigella produces effectors to down-regulate inflammation and the innate immune response. This promotes infection and limits the adaptive immune response, causing the host to remain partially susceptible to re-infection. Combinations of these virulence factors may contribute to the different symptoms and infection capabilities of the diverse Shigella species, in addition to distinct transmission patterns. Further investigation of the dominant species causing disease, using whole-genome sequencing and genotyping, will allow comparison and identification of crucial virulence factors and may contribute to the production of a pan-Shigella vaccine.
Assuntos
Disenteria Bacilar/microbiologia , Shigella/fisiologia , Fatores de Virulência/genética , Imunidade Adaptativa , Animais , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Disenteria Bacilar/imunologia , Disenteria Bacilar/metabolismo , Epitélio/imunologia , Epitélio/metabolismo , Epitélio/microbiologia , Epitélio/patologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Imunomodulação , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Plasmídeos/genética , Sistemas de Secreção Tipo III , Virulência , Fatores de Virulência/metabolismoRESUMO
Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.
Assuntos
Sistemas de Secreção Bacterianos/metabolismo , Shigella flexneri/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Mutação , Shigella flexneri/genéticaRESUMO
Type III secretion systems (T3SSs) are essential devices in the virulence of many Gram-negative bacterial pathogens. They mediate injection of protein effectors of virulence from bacteria into eukaryotic host cells to manipulate them during infection. T3SSs involved in virulence (vT3SSs) are evolutionarily related to bacterial flagellar protein export apparatuses (fT3SSs), which are essential for flagellar assembly and cell motility. The structure of the external and transmembrane parts of both fT3SS and vT3SS is increasingly well-defined. However, the arrangement of their cytoplasmic and inner membrane export apparatuses is much less clear. Here we compare the architecture of the cytoplasmic regions of the vT3SSs of Shigella flexneri and the vT3SS and fT3SS of Salmonella enterica serovar Typhimurium at ~5 and ~4 nm resolution using electron cryotomography and subtomogram averaging. We show that the cytoplasmic regions of vT3SSs display conserved six-fold symmetric features including pods, linkers and an ATPase complex, while fT3SSs probably only display six-fold symmetry in their ATPase region. We also identify other morphological differences between vT3SSs and fT3SSs, such as relative disposition of their inner membrane-attached export platform, C-ring/pods and ATPase complex. Finally, using classification, we find that both types of apparatuses can loose elements of their cytoplasmic region, which may therefore be dynamic.
RESUMO
The accessory Sec system in Streptococcus gordonii DL1 is a specialized export system that transports a large serine-rich repeat protein, Hsa, to the bacterial surface. The system is composed of core proteins SecA2 and SecY2 and accessory Sec proteins Asp1-Asp5. Similar to canonical SecYEG, SecY2 forms a channel for translocation of the Hsa adhesin across the cytoplasmic membrane. Accessory Sec proteins Asp4 and Asp5 have been suggested to work alongside SecY2 to form the translocon, similar to the associated SecY, SecE, and SecG of the canonical system (SecYEG). To test this theory, S. gordonii secY2, asp4, and asp5 were co-expressed in Escherichia coli The resultant complex was subsequently purified, and its composition was confirmed by mass spectrometry to be SecY2-Asp4-Asp5. Like SecYEG, the non-canonical complex activates the ATPase activity of the SecA motor (SecA2). This study also shows that Asp4 and Asp5 are necessary for optimal adhesion of S. gordonii to glycoproteins gp340 and fibronectin, known Hsa binding partners, as well as for early stage biofilm formation. This work opens new avenues for understanding the structure and function of the accessory Sec system.
Assuntos
Proteínas de Bactérias , Canais de Translocação SEC , Streptococcus gordonii , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA , Humanos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Canais de Translocação SEC/química , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Streptococcus gordonii/química , Streptococcus gordonii/genética , Streptococcus gordonii/metabolismo , Proteínas Supressoras de TumorRESUMO
Type III secretion systems (T3SSs) are central virulence devices for many Gram-negative bacterial pathogens of humans, animals & plants. Upon physical contact with eukaryotic host cells, they translocate virulence-mediating proteins, known as effectors, into them during infection. T3SSs are gated from the outside by host-cell contact and from the inside via two cytoplasmic negative regulators, MxiC and IpaD in Shigella flexneri, which together control the effector secretion hierarchy. Their absence leads to premature and increased secretion of effectors. Here, we investigated where and how these regulators act. We demonstrate that the T3SS inner membrane export apparatus protein MxiA plays a role in substrate selection. Indeed, using a genetic screen, we identified two amino acids located on the surface of MxiA's cytoplasmic region (MxiAC) which, when mutated, upregulate late effector expression and, in the case of MxiAI674V, also secretion. The cytoplasmic region of MxiA, but not MxiAN373D and MxiAI674V, interacts directly with the C-terminus of MxiC in a two-hybrid assay. Efficient T3S requires a cytoplasmic ATPase and the proton motive force (PMF), which is composed of the ΔΨ and the ΔpH. MxiA family proteins and their regulators are implicated in utilization of the PMF for protein export. However, our MxiA point mutants show similar PMF utilisation to wild-type, requiring primarily the ΔΨ. On the other hand, lack of MxiC or IpaD, renders the faster T3S seen increasingly dependent on the ΔpH. Therefore, MxiA, MxiC and IpaD act together to regulate substrate selection and secretion mode in the T3SS of Shigella flexneri.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Shigella flexneri/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Força Próton-Motriz , Especificidade por Substrato , Técnicas do Sistema de Duplo-HíbridoRESUMO
T3SSs are essential virulence determinants of many Gram-negative bacteria, used to inject bacterial effectors of virulence into eukaryotic host cells. Their major extracellular portion, a â¼50 nm hollow, needle-like structure, is essential to host cell sensing and the conduit for effector secretion. It is formed of a small, conserved subunit arranged as a helical polymer. The structure of the subunit has been studied by electron cryomicroscopy within native polymers and by solid-state NMR in recombinant polymers, yielding two incompatible atomic models. To resolve this controversy, we re-examined the native polymer used for electron cryomicroscopy via surface labelling and solid-state NMR. Our data show the orientation and overall fold of the subunit within this polymer is as established by solid-state NMR for recombinant polymers.
Assuntos
Proteínas de Bactérias/genética , Dobramento de Proteína , Shigella flexneri/patogenicidade , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de ProteínaRESUMO
Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at â¼ 20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.
Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos , Cisteína/metabolismo , Shigella flexneri/metabolismo , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Reagentes de Ligações Cruzadas/metabolismo , Imageamento Tridimensional , Microscopia Eletrônica , Modelos Moleculares , Simulação de Acoplamento Molecular , Multimerização Proteica , Estrutura Secundária de Proteína , Shigella flexneri/química , Shigella flexneri/genéticaRESUMO
Difficulties associated with using X-ray crystallography for structural studies of large macromolecular complexes have made single particle cryo-electron microscopy (cryoEM) a key technique in structural biology. The efficient application of the single particle cryoEM approach requires the sample to be vitrified within the holes of carbon films, with particles well dispersed throughout the ice and adopting multiple orientations. To achieve this, the carbon support film is first hydrophilised by glow discharge, which allows the sample to spread over the film. Unfortunately, for transmembrane complexes especially, this procedure can result in severe sample adsorption to the carbon support film, reducing the number of particles dispersed in the ice. This problem is rate-limiting in the single particle cryoEM approach and has hindered its widespread application to hydrophobic complexes. We describe a novel grid preparation technique that allows for good particle dispersion in the ice and minimal hydrophobic particle adhesion to the support film. This is achieved by hydrophilisation of the carbon support film by the use of selected detergents that interact with the support so as to achieve a hydrophilic and neutral or selectively charged surface.
Assuntos
Carbono/química , Microscopia Crioeletrônica/métodos , Substâncias Macromoleculares/química , Cristalografia por Raios X , VitrificaçãoRESUMO
Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB((P28T)) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB((P28T)) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the F(O)F(1)-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification.
Assuntos
Antibacterianos/farmacologia , Flagelos/metabolismo , Hidrazinas/farmacologia , Ácido Salicílico/farmacologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética , Alelos , Proteínas de Bactérias/metabolismo , Cromossomos/ultraestrutura , Farmacorresistência Bacteriana/genética , Flagelos/efeitos dos fármacos , Deleção de Genes , Movimento , Mutação , Plasmídeos/metabolismoRESUMO
Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram-negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaD's C-terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Regulação Bacteriana da Expressão Gênica , Shigella flexneri/patogenicidade , Transdução de Sinais , Fatores de Virulência/metabolismo , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Análise Mutacional de DNA , Modelos Moleculares , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Shigella flexneri/genética , Shigella flexneri/metabolismoRESUMO
The type III secretion apparatus (T3SA), which is evolutionarily and structurally related to the bacterial flagellar hook basal body, is a key virulence factor used by many gram-negative bacteria to inject effector proteins into host cells. A hollow extracellular needle forms the injection conduit of the T3SA. Its length is tightly controlled to match specific structures at the bacterial and host-cell surfaces but how this occurs remains incompletely understood. The needle is topped by a tip complex, which senses the host cell and inserts as a translocation pore in the host membrane when secretion is activated. The interaction of two conserved proteins, inner-membrane Spa40 and secreted Spa32, respectively, in Shigella, is proposed to regulate needle length and to flick a type III secretion substrate specificity switch from needle components/Spa32 to translocator/effector substrates. We found that, as in T3SAs from other species, substitution N257A within the conserved cytoplasmic NPTH region in Spa40 prevented its autocleavage and substrate specificity switching. Yet, the spa40(N257A) mutant made only slightly longer needles with a few needle tip complexes, although it could not form translocation pores. On the other hand, Δspa32, which makes extremely long needles and also formed only few tip complexes, could still form some translocation pores, indicating that it could switch substrate specificity to some extent. Therefore, loss of needle length control and defects in secretion specificity switching are not tightly coupled in either a Δspa32 mutant or a spa40(N257A) mutant.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Substâncias Macromoleculares/metabolismo , Shigella/metabolismo , Humanos , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Especificidade por SubstratoRESUMO
Type III secretion systems of Gram-negative bacteria form injection devices that deliver effector proteins into eukaryotic cells during infection. They span both bacterial membranes and the extracellular space to connect with the host cell plasma membrane. Their extracellular portion is a needle-like, hollow tube that serves as a secretion conduit for effector proteins. The needle of Shigella flexneri is approximately 50-nm long and 7-nm thick and is made by the helical assembly of one protein, MxiH. We provide a 7-Å resolution 3D image reconstruction of the Shigella needle by electron cryomicroscopy, which resolves α-helices and a ß-hairpin that has never been observed in the crystal and solution structures of needle proteins, including MxiH. An atomic model of the needle based on the 3D-density map, in comparison with that of the bacterial-flagellar filament, provides insights into how such a thin tubular structure is stably assembled by intricate intermolecular interactions. The map also illuminates how the needle-length control protein functions as a ruler within the central channel during export of MxiH for assembly at the distal end of the needle, and how the secretion-activation signal may be transduced through a conformational change of the needle upon host-cell contact.
Assuntos
Proteínas de Bactérias/química , Shigella flexneri/metabolismo , Proteínas de Bactérias/fisiologia , Calibragem , Membrana Celular/metabolismo , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Conformação Molecular , Polímeros/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais , TemperaturaRESUMO
MxiG is a single-pass membrane protein that oligomerizes within the inner membrane ring of the Shigella flexneri type III secretion system (T3SS). The MxiG N-terminal domain (MxiG-N) is the predominant cytoplasmic structure; however, its role in T3SS assembly and secretion is largely uncharacterized. We have determined the solution structure of MxiG-N residues 6-112 (MxiG-N(6-112)), representing the first published structure of this T3SS domain. The structure shows strong structural homology to forkhead-associated (FHA) domains. Canonically, these cell-signaling modules bind phosphothreonine (Thr(P)) via highly conserved residues. However, the putative phosphate-binding pocket of MxiG-N(6-112) does not align with other FHA domain structures or interact with Thr(P). Furthermore, mutagenesis of potential phosphate-binding residues has no effect on S. flexneri T3SS assembly and function. Therefore, MxiG-N has a novel function for an FHA domain. Positioning of MxiG-N(6-112) within the EM density of the S. flexneri needle complex gives insight into the ambiguous stoichiometry of the T3SS, supporting models with 24 MxiG subunits in the inner membrane ring.
Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana/química , Shigella flexneri/metabolismo , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Clonagem Molecular , Vermelho Congo/farmacologia , Sequência Conservada , Corantes Fluorescentes/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/fisiologia , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação , Fosfatos/química , Fosfotreonina/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacteria, including animal and plant pathogens. They inject 'effector' proteins through a 'needle' protruding from the bacterial surface directly into eukaryotic cells after assembly of a 'translocator' pore in the host plasma membrane. Secretion is a tightly regulated process, which is blocked until physical contact with a host cell takes place. Host cell sensing occurs through a distal needle 'tip complex' and translocators are secreted before effectors. MxiC, a Shigella T3SS substrate, prevents premature effector secretion. Here, we examine how the different parts of T3SSs work together to allow orderly secretion. We show that T3SS assembly and needle tip composition are not altered in an mxiC mutant. We find that MxiC not only represses effector secretion but that it is also required for translocator release. We provide genetic evidence that MxiC acts downstream of the tip complex and then the needle during secretion activation. Finally, we show that the needle controls MxiC release. Therefore, for the first time, our data allow us to propose a model of secretion activation that goes from the tip complex to cytoplasmic MxiC via the needle.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Disenteria Bacilar/microbiologia , Interações Hospedeiro-Patógeno , Shigella flexneri/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Transporte Proteico , Shigella flexneri/genéticaRESUMO
Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacterial pathogens. Upon cell contact, they inject effector proteins directly into eukaryotic cells through a needle protruding from the bacterial surface. Host cell sensing occurs through a distal needle "tip complex," but how this occurs is not understood. The tip complex of quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. It binds needles via its extreme C-terminal coiled coil, thereby likely positioning a large domain containing its hydrophobic regions at the distal tips of needles. In this study, we used short deletion mutants within this domain to search for regions of IpaB involved in secretion regulation. This identified two regions, amino acids 227 to 236 and 297 to 306, the presence of which are required for maintenance of IpaB at the needle tip, secretion regulation, and normal pore formation but not invasion. We therefore propose that removal of either of these regions leads to an inability to block secretion prior to reception of the activation signal and/or a defect in host cell sensing.
Assuntos
Proteínas de Bactérias/fisiologia , Sistemas de Secreção Bacterianos/fisiologia , Disenteria Bacilar/microbiologia , Shigella flexneri/patogenicidade , Antígenos de Bactérias/genética , Antígenos de Bactérias/fisiologia , Aderência Bacteriana/fisiologia , Sistemas de Secreção Bacterianos/genética , Membrana Eritrocítica/microbiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Células HeLa , Humanos , Microscopia de Fluorescência , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Deleção de Sequência/genética , Shigella flexneri/genéticaRESUMO
Type III secretion systems (T3SSs) are widely distributed virulence determinants of Gram-negative bacteria. They translocate bacterial proteins into host cells to manipulate them during infection. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region, and a hollow needle protruding from the bacterial surface. The distal tip of mature, quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. However, its mode of needle association is unknown. Here, we show that deletion of 3 or 9 residues at the C terminus of IpaB leads to fast constitutive secretion of late effectors, as observed in a DeltaipaB strain. Like the DeltaipaB mutant, mutants with C-terminal mutations also display hyperadhesion. However, unlike the DeltaipaB mutant, they are still invasive and able to lyse the internalization vacuole with nearly wild-type efficiency. Finally, the mutant proteins show decreased association with needles and increased recruitment of IpaC. Taken together, these data support the notion that the state of the tip complex regulates secretion. We propose a model where the quiescent needle tip has an "off" conformation that turns "on" upon host cell contact. Our mutants may adopt a partially "on" conformation that activates secretion and is capable of recruiting some IpaC to insert pores into host cell membranes and allow invasion.