RESUMO
Bacterial pathogens utilize the factors of their hosts to infect them, but which factors they exploit remain poorly defined. Here, we show that a pathogenic Salmonella enterica serovar Typhimurium (STm) exploits host polyamines for the functional expression of virulence factors. An STm mutant strain lacking principal genes required for polyamine synthesis and transport exhibited impaired infectivity in mice. A polyamine uptake-impaired strain of STm was unable to inject effectors of the type 3 secretion system into host cells due to a failure of needle assembly. STm infection stimulated host polyamine production by increasing arginase expression. The decline in polyamine levels caused by difluoromethylornithine, which inhibits host polyamine production, attenuated STm colonization, whereas polyamine supplementation augmented STm pathogenesis. Our work reveals that host polyamines are a key factor promoting STm infection, and therefore a promising therapeutic target for bacterial infection.
Assuntos
Poliaminas , Salmonella typhimurium , Sistemas de Secreção Tipo III , Fatores de Virulência , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/genética , Animais , Poliaminas/metabolismo , Camundongos , Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo III/genética , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno , Humanos , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , FemininoRESUMO
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common foodborne enteric pathogen that infects humans or mammals and colonizes the intestinal tract primarily by invading the host following ingestion. Meanwhile, ClpV is a core secreted protein of the bacterial type VI secretion system (T6SS). Because elucidating ClpV's role in the pathogenesis of T6SS is pivotal for revealing the virulence mechanism of Salmonella, in our study, clpV gene deletion mutants were constructed using a λ-red-based recombination system, and the effect of clpV mutation on SL1344's pathogenicity was examined in terms of stress resistance, motility, cytokine secretion, gut microbiota, and a BALB/c mouse model. Among the results, ClpV affected SL1344's motility and was also involved in cell invasion, adhesion, and intracellular survival in the MDBK cell model but did not affect invasion or intracellular survival in the RAW264.7 cell model. Moreover, clpV gene deletion significantly reduced the transcription levels of GBP2b, IFNB1, IL-6, NLRP3, NOS2, and TNF-α proinflammatory factor levels but significantly increased transcription levels of IL-4 and IL-10 anti-inflammatory factors. Last, ClpV appeared to closely relate to the pathogenicity of S. Typhimurium in vivo, which can change the gut environment and cause dysbiosis of gut microbiota. Our findings elucidate the functions of ClpV in S. Typhimurium and illustrating interactions between T6SS and gut microbiota help to clarify the mechanisms of the pathogenesis of foodborne diseases.
Assuntos
Proteínas de Bactérias , Microbioma Gastrointestinal , Camundongos Endogâmicos BALB C , Salmonella typhimurium , Animais , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/genética , Camundongos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Virulência , Humanos , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Feminino , Infecções por Salmonella/microbiologia , Infecções por Salmonella/imunologia , Células RAW 264.7RESUMO
Invasive non-typhoidal Salmonella (iNTS) disease is an under-recognized high-burden disease causing major health and socioeconomic issues in sub-Saharan Africa (sSA), predominantly among immune-naïve infants and young children, including those with recognized comorbidities such as HIV infection. iNTS disease is primarily caused by Salmonella enterica serovar Typhimurium sequence type (ST) 313 and 'African-restricted clades' of Salmonella Enteritidis ST11 that have emerged across the African continent as a series of epidemics associated with acquisition of new antimicrobial resistance. Due to genotypes with a high prevalence of antimicrobial resistance and scarcity of therapeutic options, these NTS serovars are designated by the World Health Organization as a priority pathogen for research and development of interventions, including vaccines, to address and reduce NTS associated bacteremia and meningitis in sSA. Novel and traditional vaccine technologies are being applied to develop vaccines against iNTS disease, and the results of the first clinical trials in the infant target population should become available in the near future. The "Vaccine Value Profile" (VVP) addresses information related predominantly to invasive disease caused by Salmonella Enteritidis and Salmonella Typhimurium prevalent in sSA. Information is included on stand-alone iNTS disease candidate vaccines and candidate vaccines targeting iNTS disease combined with another invasive serotype, Salmonella Typhi, that is also common across sSA. Out of scope for the first version of this VVP is a wider discussion on either diarrheagenic NTS disease (dNTS) also associated with Salmonella Enteritidis and Salmonella Typhimurium or the development of a multivalent Salmonella vaccines targeting key serovars for use globally. This VVP for vaccines to prevent iNTS disease is intended to provide a high-level, holistic assessment of the information and data that are currently available to inform the potential public health, economic, and societal value of pipeline vaccines and vaccine-like products. Future versions of this VVP will be updated to reflect ongoing activities such as vaccine development strategies and a "Full Vaccine Value Assessment" that will inform the value proposition of an iNTS disease vaccine. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public private partnerships, and multi-lateral organizations, and in collaboration with stakeholders from the World Health Organization African Region. All contributors have extensive expertise on various elements of the iNTS disease VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information.
Assuntos
Infecções por Salmonella , Vacinas contra Salmonella , Salmonella enteritidis , Humanos , África Subsaariana/epidemiologia , Salmonella enteritidis/imunologia , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Infecções por Salmonella/prevenção & controle , Infecções por Salmonella/epidemiologia , Infecções por Salmonella/microbiologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/genética , Vacinas contra Salmonella/imunologia , Vacinas contra Salmonella/administração & dosagemRESUMO
In order to survive and replicate, Salmonella has evolved mechanisms to gain access to intestinal epithelial cells of the crypt. However, the impact of Salmonella Typhimurium on stem cells and progenitors, which are responsible for the ability of the intestinal epithelium to renew and protect itself, remains unclear. Given that intestinal organoids growth is sustained by stem cells and progenitors activity, we have used this model to document the effects of Salmonella Typhimurium infection on epithelial proliferation and differentiation, and compared it to an in vivo model of Salmonella infection in mice. Among gut segments, the caecum was preferentially targeted by Salmonella. Analysis of infected crypts and organoids demonstrated increased length and size, respectively. mRNA transcription profiles of infected crypts and organoids pointed to upregulated EGFR-dependent signals, associated with a decrease in secretory cell lineage differentiation. To conclude, we show that organoids are suited to mimic the impact of Salmonella on stem cells and progenitors cells, carrying a great potential to drastically reduce the use of animals for scientific studies on that topic. In both models, the EGFR pathway, crucial to stem cells and progenitors proliferation and differentiation, is dysregulated by Salmonella, suggesting that repeated infections might have consequences on crypt integrity and further oncogenesis.
Assuntos
Diferenciação Celular , Receptores ErbB , Organoides , Infecções por Salmonella , Salmonella typhimurium , Células-Tronco , Animais , Organoides/microbiologia , Células-Tronco/metabolismo , Camundongos , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Receptores ErbB/metabolismo , Receptores ErbB/genética , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Proliferação de Células , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
Infection of ruminants such as cattle with Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a disease characterized by chronic inflammation of the small intestine and diarrhoea. Infection with MAP is acquired via the faecal-to-oral route and the pathogen initially invades the epithelial lining of the small intestine. In this study we used an in vitro 3D mouse enteroid model to determine the influence of M cells in infection of the gut epithelia by MAP, in comparison with another bacterial intestinal pathogen of veterinary importance, Salmonella enterica serovar Typhimurium. The differentiation of M cells in the enteroid cultures was induced by stimulation with the cytokine receptor activator of nuclear factor-κB ligand (RANKL), and the effects on MAP and Salmonella uptake and intracellular survival were determined. The presence of M cells in the cultures correlated with increased uptake and intracellular survival of Salmonella, but had no effect on MAP. Interestingly neither pathogen was observed to preferentially accumulate within GP2-positive M cells.
Assuntos
Mycobacterium avium subsp. paratuberculosis , Salmonella typhimurium , Animais , Mycobacterium avium subsp. paratuberculosis/fisiologia , Salmonella typhimurium/fisiologia , Salmonella typhimurium/patogenicidade , Camundongos , Paratuberculose/microbiologia , Viabilidade Microbiana , Mucosa Intestinal/microbiologia , Bovinos , Células MRESUMO
The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterize the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica serovar Enteritidis that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. sseM is widely distributed among the five subspecies of Salmonella enterica, is found in many clinically relevant serovars, and is co-transcribed with pipB2, a SPI-2 effector gene. The translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely, ß-2-syntrophin, utrophin/dystrophin, α-catulin, α-dystrobrevin, and ß-dystrobrevin. The interaction between SseM and ß-2-syntrophin and α-dystrobrevin was verified in Salmonella Typhimurium-infected cells and relied on the postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain of ß-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A ΔsseM mutant strain had a small competitive advantage over the wild-type strain in the S. Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild-type SseM from S. Typhimurium or S. Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates the systemic growth of bacteria in mice. Furthermore, the ΔsseM mutant strain displayed enhanced replication in bone marrow-derived macrophages, demonstrating that SseM restrains intracellular bacterial growth to modulate Salmonella virulence. IMPORTANCE: In Salmonella enterica, the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells, which are required for Salmonella virulence. The identification and functional characterization of SPI-2 injectisome effectors advance our understanding of the interplay between Salmonella and its host(s). Using an optimized method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal Salmonella enterica serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector-SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif that is essential for interaction with the PDZ-containing host protein ß-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.
Assuntos
Proteínas de Bactérias , Salmonella enteritidis , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Humanos , Camundongos , Virulência , Salmonella enteritidis/genética , Salmonella enteritidis/metabolismo , Salmonella enteritidis/patogenicidade , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Infecções por Salmonella/microbiologia , Proteínas Associadas à Distrofina/metabolismo , Proteínas Associadas à Distrofina/genética , Ilhas Genômicas , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Proteômica , Modelos Animais de Doenças , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
Host-generated antimicrobial peptides (AMPs) play a pivotal role in defense against bacterial pathogens. AMPs kill invading bacteria majorly by disrupting the bacterial cell walls. AMPs are actively synthesized and released into the lumen of the gastrointestinal tract to limit colonization of enteric pathogens like Salmonella typhimurium (S. typhimurium). However, S. typhimurium has evolved several resistance mechanisms to defend AMPs. The multicomponent SapABCDF uptake transporter is one such system that helps in resisting AMPs. In the current study, we analyzed the role of S. typhimurium SapA against stress survival and virulence of this bacterium. ∆sapA mutant strain showed hypersensitivity to AMPs, like melittin and mastoparan. Further, ∆sapA mutant showed more than 22 folds (p = 0.019) hypersensitivity to neutrophils as compared to the WT strain of S. typhimurium. In addition, ∆sapA strain showed defective survival in mice. In conclusion, the results of the current study suggest that the SapA is essential for survival against AMPs and virulence of S. typhimurium.
Assuntos
Neutrófilos , Salmonella typhimurium , Animais , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/genética , Camundongos , Neutrófilos/imunologia , Neutrófilos/microbiologia , Virulência , Peptídeos Antimicrobianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Camundongos Endogâmicos BALB C , Infecções por Salmonella/microbiologia , Peptídeos Catiônicos Antimicrobianos/farmacologiaRESUMO
To study potential ramifications of antimicrobial resistance, we carried out adaptive laboratory evolution assays (ALE) to isolate three resistant variants (RVs) of Salmonella enterica Typhimurium, employing three different types of food preservation methods: 1) an emergent technology, plasma-activated water (PAW), leading to variant RV-PAW; a traditional method, heat, leading to variant RV-HT, and a natural antimicrobial compound, carvacrol, leading to variant RV-CAR. The variant resistant to plasma-activated water, RV-PAW, had mutations in rpoA and rpoD; it showed increased tolerance to heat in orange juice but ultimately did not pose a significant threat, as it exhibited a fitness cost at refrigeration temperature (8 °C), whereas its virulence against Caenorhabditis elegans decreased. The variant resistant to heat, RV-HT, had mutations in flhC, dnaJ: it exhibited a fitness cost at high growth temperatures (43 °C) and induced morphofunctional alterations in C. elegans. The variant resistant to carvacrol, RV-CAR, had mutations in sseG, flhA, wbaV, lon; this variant not only exhibited significantly higher thermotolerance in both laboratory media and food models but also effectively increased its growth fitness at refrigeration temperatures while retaining its virulence, evidenced by the highest percentage of Smurf phenotype in C. elegans. To address these challenges, we applied a process combining thermal treatment with citral, with the aim of leveraging the sublethal damage caused in RVs by heat treatments in orange juice. This approach achieves enhanced microbial inactivation without having to escalate the intensity of the thermal treatment. The result was particularly encouraging in the case of RV-CAR, the most challenging strain, for which we improved lethality by up to 3 log10 inactivation cycles.
Assuntos
Caenorhabditis elegans , Conservação de Alimentos , Temperatura Alta , Salmonella typhimurium , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/patogenicidade , Virulência , Caenorhabditis elegans/microbiologia , Animais , Conservação de Alimentos/métodos , Termotolerância , Mutação , Microbiologia de Alimentos , Farmacorresistência Bacteriana/genética , Cimenos/farmacologiaRESUMO
Lysozyme is a ß-1,4-glycosidase that hydrolyzes the polysaccharide backbone of bacterial cell walls. With an additional bactericidal function mediated by a separate protein domain, lysozyme is considered a uniquely important antimicrobial molecule contributing to the host's innate immune response to infection. Elevated lysozyme production is found in various inflammatory conditions while patients with genetic risks for inflammatory bowel diseases demonstrate abnormal lysozyme expression, granule packaging, and secretion in Paneth cells. However, it remains unclear how a gain- or loss-of-function in host lysozyme may impact the host inflammatory responses to pathogenic infection. We challenged Lyz1-/- and ectopic Lyz1-expressing (Villin-Lyz1TG) mice with S. Typhimurium and then comprehensively assessed the inflammatory disease progression. We conducted proteomics analysis to identify molecules derived from human lysozyme-mediated processing of live Salmonella. We examined the barrier-impairing effects of these identified molecules in human intestinal epithelial cell monolayer and enteroids. Lyz1-/- mice are protected from infection in terms of morbidity, mortality, and barrier integrity, whereas Villin-Lyz1TG mice demonstrate exacerbated infection and inflammation. The growth and invasion of Salmonella in vitro are not affected by human or chicken lysozyme, whereas lysozyme encountering of live Salmonella stimulates the release of barrier-disrupting factors, InvE-sipC and Lpp1, which directly or indirectly impair the tight junctions. The direct engagement of host intestinal lysozyme with an enteric pathogen such as Salmonella promotes the release of virulence factors that are barrier-impairing and pro-inflammatory. Controlling lysozyme function may help alleviate the inflammatory progression.
Assuntos
Muramidase , Salmonella typhimurium , Muramidase/metabolismo , Animais , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Camundongos , Humanos , Infecções por Salmonella/microbiologia , Infecções por Salmonella/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Camundongos Knockout , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas dos MicrofilamentosRESUMO
The gastrointestinal tract is densely colonized by a polymicrobial community known as the microbiota which serves as primary line of defence against pathogen invasion. The microbiota can limit gut-luminal pathogen growth at different stages of infection. This can be traced to specific commensal strains exhibiting direct or indirect protective functions. Although these mechanisms hold the potential to develop new approaches to combat enteric pathogens, they remain far from being completely described. In this study, we investigated how a mouse commensal Escherichia coli can outcompete Salmonella enterica serovar Typhimurium (S. Tm). Using a salmonellosis mouse model, we found that the commensal E. coli 8178 strain relies on a trojan horse trap strategy to limit S. Tm expansion in the inflamed gut. Combining mutants and reporter tools, we demonstrated that inflammation triggers the expression of the E. coli 8178 antimicrobial microcin H47 toxin which, when fused to salmochelin siderophores, can specifically alter S. Tm growth. This protective function was compromised upon disruption of the E. coli 8178 tonB-dependent catecholate siderophore uptake system, highlighting a previously unappreciated crosstalk between iron intake and microcin H47 activity. By identifying the genetic determinants mediating S. Tm competition, our work not only provides a better mechanistic understanding of the protective function displayed by members of the gut microbiota but also further expands the general contribution of microcins in bacterial antagonistic relationships. Ultimately, such insights can open new avenues for developing microbiota-based approaches to better control intestinal infections.
Assuntos
Escherichia coli , Inflamação , Salmonella typhimurium , Sideróforos , Animais , Escherichia coli/metabolismo , Escherichia coli/genética , Sideróforos/metabolismo , Camundongos , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/metabolismo , Inflamação/metabolismo , Inflamação/microbiologia , Camundongos Endogâmicos C57BL , Bacteriocinas/metabolismo , Bacteriocinas/farmacologia , Microbioma Gastrointestinal , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Infecções por Salmonella/microbiologia , Infecções por Salmonella/metabolismo , Feminino , Ferro/metabolismo , Simbiose , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/metabolismoRESUMO
Natural killer (NK) cells play a key role in defense against Salmonella infections during the early phase of infection. Our previous work showed that the excretory/secretory products of Ascaris suum repressed NK activity in vitro. Here, we asked if NK cell functionality was influenced in domestic pigs during coinfection with Ascaris and Salmonella enterica serotype Typhimurium. Ascaris coinfection completely abolished the IL-12 and IL-18 driven elevation of IFN-γ production seen in CD16 + CD8α + perforin + NK cells of Salmonella single-infected pigs. Furthermore, Ascaris coinfection prohibited the Salmonella-driven rise in NK perforin levels and CD107a surface expression. In line with impaired effector functions, NK cells from Ascaris-single and coinfected pigs displayed elevated expression of the inhibitory KLRA1 and NKG2A receptors genes, contrasting with the higher expression of the activating NKp46 and NKp30 receptors in NK cells during Salmonella single infection. These differences were accompanied by the highly significant upregulation of T-bet protein expression in NK cells from Ascaris-single and Ascaris/Salmonella coinfected pigs. Together, our data strongly indicate a profound repression of NK functionality by an Ascaris infection which may hinder infected individuals from adequately responding to a concurrent bacterial infection.
Assuntos
Ascaríase , Coinfecção , Células Matadoras Naturais , Doenças dos Suínos , Animais , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ascaríase/imunologia , Ascaríase/veterinária , Ascaríase/parasitologia , Coinfecção/imunologia , Coinfecção/microbiologia , Coinfecção/parasitologia , Suínos , Doenças dos Suínos/parasitologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Salmonelose Animal/imunologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Ascaris suum/imunologia , Interferon gama/metabolismo , Perforina/metabolismo , Interleucina-12/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/genética , Interleucina-18/metabolismoRESUMO
Mass spectrometry-based proteomics provides a wealth of information about changes in protein production and abundance under diverse conditions, as well as mechanisms of regulation, signaling cascades, interaction partners, and communication patterns across biological systems. For profiling of intracellular pathogens, proteomic profiling can be performed in the absence of a host to singularly define the pathogenic proteome or during an infection-like setting to identify dual perspectives of infection. In this chapter, we present techniques to extract proteins from the human bacterial intracellular pathogen, Salmonella enterica serovar Typhimurium, in the presence of macrophages, an important innate immune cell in host defense. We outline sample preparation, including protein extraction, digestion, and purification, as well as mass spectrometry measurements and bioinformatics analysis. The data generated from our dual perspective profiling approach provides new insight into pathogen and host protein modulation under infection-like conditions.
Assuntos
Proteínas de Bactérias , Macrófagos , Proteômica , Salmonella typhimurium , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Proteômica/métodos , Humanos , Proteínas de Bactérias/metabolismo , Macrófagos/microbiologia , Macrófagos/metabolismo , Interações Hospedeiro-Patógeno , Proteoma/metabolismo , Infecções por Salmonella/microbiologia , Infecções por Salmonella/metabolismo , Infecções por Salmonella/imunologia , Biologia Computacional/métodos , Espectrometria de Massas/métodosRESUMO
Salmonella enterica subspecies enterica serovar Typhimurium is an intracellular pathogen that invades and colonizes the intestinal epithelium. Following bacterial invasion, Salmonella is enclosed within a membrane-bound vacuole known as a Salmonella-containing vacuole (SCV). However, a subset of Salmonella has the capability to prematurely rupture the SCV and escape, resulting in Salmonella hyper-replication within the cytosol of epithelial cells. A recently published RNA-seq study provides an overview of cytosolic and vacuolar upregulated genes and highlights pagN vacuolar upregulation. Here, using transcription kinetics, protein production profile, and immunofluorescence microscopy, we showed that PagN is exclusively produced by Salmonella in SCV. Gentamicin protection and chloroquine resistance assays were performed to demonstrate that deletion of pagN affects Salmonella replication by affecting the cytosolic bacterial population. This study presents the first example of a Salmonella virulence factor expressed within the endocytic compartment, which has a significant impact on the dynamics of Salmonella cytosolic hyper-replication.
Assuntos
Proteínas de Bactérias , Citosol , Salmonella typhimurium , Vacúolos , Fatores de Virulência , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Citosol/microbiologia , Vacúolos/microbiologia , Vacúolos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Humanos , Virulência , Infecções por Salmonella/microbiologia , Células HeLa , Células Epiteliais/microbiologia , Regulação Bacteriana da Expressão GênicaRESUMO
Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.
Assuntos
Proteínas de Ligação ao Cálcio , Citosol , Flagelina , Interações Hospedeiro-Patógeno , Inflamassomos , Salmonella typhimurium , Sistemas de Secreção Tipo III , Citosol/metabolismo , Citosol/microbiologia , Animais , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Inflamassomos/metabolismo , Camundongos , Flagelina/metabolismo , Proteína Inibidora de Apoptose Neuronal/metabolismo , Proteína Inibidora de Apoptose Neuronal/genética , Células Epiteliais/microbiologia , Células Epiteliais/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Camundongos Endogâmicos C57BL , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Análise de Célula Única/métodos , Infecções por Salmonella/microbiologia , Infecções por Salmonella/metabolismo , Infecções por Salmonella/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/metabolismoRESUMO
Intestinal edema is a common manifestation of numerous gastrointestinal diseases and is characterized by the accumulation of fluid in the interstitial space of the intestinal wall. Technical advances in laser capture microdissection and low-biomass proteomics now allow us to specifically characterize the intestinal edema proteome. Using advanced proteomics, we identify peptides derived from antimicrobial factors with high signal intensity, but also highlight major contributions from the blood clotting system, extracellular matrix (ECM) and protease-protease inhibitor networks. The ECM is a complex fibrillar network of macromolecules that provides structural and mechanical support to the intestinal tissue. One abundant component of the ECM observed in Salmonella-driven intestinal edema is the glycoprotein fibronectin, recognized for its structure-function interplay regulated by mechanical forces. Using mechanosensitive staining of fibronectin fibers reveals that they are tensed in the edema, despite the high abundance of proteases able to cleave fibronectin. In contrast, fibronectin fibers increasingly relax in other cecal tissue areas as the infection progresses. Co-staining for fibrin(ogen) indicates the formation of a provisional matrix in the edema, similar to what is observed in response to skin injury, while collagen staining reveals a sparse and disrupted collagen fiber network. These observations plus the absence of low tensional fibronectin fibers and the additional finding of a high number of protease inhibitors in the edema proteome could indicate a critical role of stretched fibronectin fibers in maintaining tissue integrity in the severely inflamed cecum. Understanding these processes may also provide valuable functional diagnostic markers of intestinal disease progression in the future.
Assuntos
Edema , Fibronectinas , Animais , Fibronectinas/metabolismo , Camundongos , Edema/metabolismo , Edema/patologia , Edema/microbiologia , Matriz Extracelular/metabolismo , Proteômica/métodos , Camundongos Endogâmicos C57BL , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Infecções por Salmonella/metabolismo , Intestinos/microbiologia , Intestinos/patologia , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/metabolismoRESUMO
Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN-7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella-containing vacuoles after long-term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread.
Assuntos
Proteínas do Citoesqueleto , Salmonella typhimurium , Proteínas rho de Ligação ao GTP , Humanos , Actinas/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Citoesqueleto/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Salmonella typhimurium/patogenicidade , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismoRESUMO
The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long-chain fatty acids, which are highly abundant throughout the host intestine, directly bind to and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerization, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. Long-chain fatty acids bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerization interface. Our results highlight two distinct, mutually exclusive mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads.
Assuntos
Proteínas de Bactérias , Intestinos , Salmonella typhimurium , Proteínas de Bactérias/metabolismo , Ácidos Graxos/metabolismo , Regulação Bacteriana da Expressão Gênica , Intestinos/metabolismo , Intestinos/microbiologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Virulência , Animais , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologiaRESUMO
This study presents the engineering of a less endotoxic Salmonella Typhimurium strain by manipulating the lipid-A structure of the lipopolysaccharide (LPS) component. Salmonella lipid A was dephosphorylated by using lpxE from Francisella tularensis. The 1-phosphate group from lipid-A was removed selectively, resulting in a close analog of monophosphoryl lipid A. We observed a significant impact of ∆pagL on major virulence factors such as biofilm formation, motility, persistency, and immune evasion. In correlation with biofilm and motility retardation, adhesion and invasion were elevated but with reduced intracellular survival, a favorable phenotype prospect of a vaccine strain. Western blotting and silver staining confirmed the absence of the O-antigen and truncated lipid-A core in the detoxified Salmonella mutant. In vitro and in vivo studies demonstrated that the dephosphorylated Salmonella mutant mediated lower pro-inflammatory cytokine secretion than the wild-type strain. The vaccine strains were present in the spleen and liver for five days and were cleared from the organs by day seven. However, the wild-type strain persisted in the spleen, liver, and brain, leading to sepsis-induced death. Histological evaluations of tissue samples further confirmed the reduced endotoxic activity of the detoxified Salmonella mutant. The detoxification strategy did not compromise the level of protective immunity, as the vaccine strain could enhance humoral and cellular immune responses and protect against the wild-type challenge in immunized mice.
Assuntos
Infecções por Salmonella , Vacinas contra Salmonella , Salmonella typhimurium , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Feminino , Animais , Camundongos , Camundongos Endogâmicos BALB C , Lipídeo A/metabolismo , Vacinas contra Salmonella/efeitos adversos , Vacinas contra Salmonella/genética , Vacinas contra Salmonella/imunologia , Lipopolissacarídeos/metabolismo , Imunidade Humoral , Imunidade Celular , Biofilmes , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Hidrolases de Éster Carboxílico/genéticaRESUMO
Quantifying the effect of public health actions on population health is essential when justifying sustained public health investment. Using modeling, we conservatively estimated that rapid response to a multistate foodborne outbreak of Salmonella Typhimurium in the United States in 2018 potentially averted 94 reported cases and $633,181 in medical costs and productivity losses.
Assuntos
Saúde Pública , Saladas , Intoxicação Alimentar por Salmonella/epidemiologia , Salmonella typhimurium , Animais , Galinhas , Surtos de Doenças , Humanos , Saúde Pública/métodos , Saladas/efeitos adversos , Saladas/microbiologia , Intoxicação Alimentar por Salmonella/economia , Intoxicação Alimentar por Salmonella/etiologia , Salmonella typhimurium/isolamento & purificação , Salmonella typhimurium/patogenicidade , Estados Unidos/epidemiologiaRESUMO
The rapid identification of bacterial antibiotic susceptibility is pivotal to the rational administration of antibacterial drugs. In this study, cefotaxime (CTX)-derived resistance in Salmonella typhimurium (abbr. CTXr-S. typhimurium) during 3 months of exposure was rapidly recorded using a portable Raman spectrometer. The molecular changes that occurred in the drug-resistant strains were sensitively monitored in whole cells by label-free surface-enhanced Raman scattering (SERS). Various degrees of resistant strains could be accurately discriminated by applying multivariate statistical analyses to bacterial SERS profiles. Minimum inhibitory concentration (MIC) values showed a positive linear correlation with the relative Raman intensities of I990/I1348, and the R2 reached 0.9962. The SERS results were consistent with the data obtained by MIC assays, mutant prevention concentration (MPC) determinations, and Kirby-Bauer antibiotic susceptibility tests (K-B tests). This preliminary proof-of-concept study indicates the high potential of the SERS method to supplement the time-consuming conventional method and help alleviate the challenges of antibiotic resistance in clinical therapy.