RESUMEN
ESKAPE pathogens are responsible for complicated nosocomial infections worldwide and are often resistant to commonly used antibiotics in clinical settings. Among ESKAPE, vancomycin-resistant Enterococcus faecium (VREfm) and methicillin-resistant Staphylococcus aureus (MRSA) are two important Gram-positive pathogens for which non-antibiotic alternatives are urgently needed. We previously showed that the lipoprotein AdcA of E. faecium elicits opsonic and protective antibodies against E. faecium and E. faecalis. Prompted by our observation, reported here, that AdcA also elicits opsonic antibodies against MRSA and other clinically relevant Gram-positive pathogens, we identified the dominant epitope responsible for AdcA cross-reactive activity and designed a hyper-thermostable and multi-presenting antigen, Sc(EH)3. We demonstrate that antibodies raised against Sc(EH)3 mediate opsonic killing of a wide-spectrum of Gram-positive pathogens, including VREfm and MRSA, and confer protection both in passive and active immunisation models. Our data indicate that Sc(EH)3 is a promising antigen for the development of vaccines against different Gram-positive pathogens.
RESUMEN
As an important immune stimulator and modulator, IFNγ is crucial for gut homeostasis and its dysregulation links to diverse colon pathologies, such as colitis and colorectal cancer (CRC). Here, we demonstrated that the epigenetic regulator, CBX3 (also known as HP1γ) antagonizes IFNγ signaling in the colon epithelium by transcriptionally repressing two critical IFNγ-responsive genes: STAT1 and CD274 (encoding Programmed death-ligand 1, PD-L1). Accordingly, CBX3 deletion resulted in chronic mouse colon inflammation, accompanied by upregulated STAT1 and CD274 expressions. Chromatin immunoprecipitation indicated that CBX3 tethers to STAT1 and CD274 promoters to inhibit their expression. Reversely, IFNγ significantly reduces CBX3 binding to these promoters and primes gene expression. This antagonist effect between CBX3 and IFNγ on STAT1/PD-L1 expression was also observed in CRC. Strikingly, CBX3 deletion heightened CRC cells sensitivity to IFNγ, which ultimately enhanced their chemosensitivity under IFNγ stimulation in vitro with CRC cells and in vivo with a syngeneic mouse tumor model. Overall, this work reveals that by negatively tuning IFNγ-stimulated immune genes' transcription, CBX3 participates in modulating colon inflammatory response and CRC chemo-resistance.
Asunto(s)
Antígeno B7-H1 , Proteínas Cromosómicas no Histona , Neoplasias Colorrectales , Interferón gamma , Factor de Transcripción STAT1 , Animales , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Interferón gamma/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Humanos , Ratones , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Colitis/metabolismo , Colitis/patología , Ratones Endogámicos C57BL , Transducción de Señal , Línea Celular TumoralRESUMEN
Preterm birth is currently the leading cause of neonatal morbidity and mortality. Genetic, immunological and infectious causes are suspected. Preterm infants have a higher risk of severe bacterial neonatal infections, most of which are caused by Escherichia coli an in particular E. coli K1strains. Women with history of preterm delivery have a high risk of recurrence and therefore constitute a target population for the development of vaccine against E. coli neonatal infections. Here, we characterize the immunological, microbiological and protective properties of a live attenuated vaccine candidate in adult female mice and their pups against after a challenge by K1 and non-K1 strains of E. coli. Our results show that the E. coli K1 E11 ∆aroA vaccine induces strong immunity, driven by polyclonal bactericidal antibodies. In our model of meningitis, mothers immunized prior to mating transfer maternal antibodies to pups, which protect newborn mice against various K1 and non-K1 strains of E. coli. Given the very high mortality rate and the neurological sequalae associated with neonatal E. coli K1 meningitis, our results constitute preclinical proof of concept for the development of a live attenuated vaccine against severe E. coli infections in women at risk of preterm delivery.
Asunto(s)
Infecciones por Escherichia coli , Enfermedades del Recién Nacido , Meningitis , Nacimiento Prematuro , Lactante , Adulto , Recién Nacido , Femenino , Animales , Ratones , Humanos , Escherichia coli/genética , Vacunas Atenuadas , Nacimiento Prematuro/prevención & control , Recien Nacido Prematuro , Infecciones por Escherichia coli/prevención & control , Enfermedades del Recién Nacido/etiología , Anticuerpos , Meningitis/etiologíaRESUMEN
The increase in emerging drug resistant Gram-negative bacterial infections is a global concern. In addition, there is growing recognition that compromising the microbiota through the use of broad-spectrum antibiotics can impact long term patient outcomes. Therefore, there is the need to develop new bactericidal strategies to combat Gram-negative infections that would address these specific issues. In this study, we report and characterize one such approach, an antibody-drug conjugate (ADC) that combines (i) targeting the surface of a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of an antimicrobial peptide. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance: Pseudomonas aeruginosa. To target this organism, we designed an ADC by fusing an antimicrobial peptide to the C-terminal end of the VH and/or VL-chain of a monoclonal antibody, VSX, that targets the core of P. aeruginosa lipopolysaccharide. This ADC demonstrates appropriately minimal levels of toxicity against mammalian cells, rapidly kills P. aeruginosa strains, and protects mice from P. aeruginosa lung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study might result in a broadly useful strategy for targeting specific pathogenic microorganisms without further augmenting antibiotic resistance.
Asunto(s)
Infecciones Bacterianas , Inmunoconjugados , Animales , Ratones , Pseudomonas aeruginosa , Anticuerpos Monoclonales/farmacología , Antibacterianos/farmacología , Péptidos Antimicrobianos , MamíferosRESUMEN
BACKGROUND: Worldwide, Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but full understanding of the pathogenesis of this disease is not yet achieved. Moreover, to date, no vaccine is available against bacterial neonatal meningitis. METHODS: Here, we used Transposon Sequencing of saturated banks of mutants (TnSeq) to evaluate E. coli K1 genetic fitness in murine neonatal meningitis. We identified E. coli K1 genes encoding for factors important for systemic dissemination and brain infection, and focused on products with a likely outer-membrane or extra-cellular localization, as these are potential vaccine candidates. We used in vitro and in vivo models to study the efficacy of active and passive immunization. RESULTS: We selected for further study the conserved surface polysaccharide Poly-ß-(1-6)-N-Acetyl Glucosamine (PNAG), as a strong candidate for vaccine development. We found that PNAG was a virulence factor in our animal model. We showed that both passive and active immunization successfully prevented and/or treated meningitis caused by E. coli K1 in neonatal mice. We found an excellent opsonophagocytic killing activity of the antibodies to PNAG and in vitro these antibodies were also able to decrease binding, invasion and crossing of E. coli K1 through two blood brain barrier cell lines. Finally, to reinforce the potential of PNAG as a vaccine candidate in bacterial neonatal meningitis, we demonstrated that Group B Streptococcus, the main cause of neonatal meningitis in developed countries, also produced PNAG and that antibodies to PNAG could protect in vitro and in vivo against this major neonatal pathogen. INTERPRETATION: Altogether, these results indicate the utility of a high-throughput DNA sequencing method to identify potential immunotherapy targets for a pathogen, including in this study a potential broad-spectrum target for prevention of neonatal bacterial infections. FUNDINGS: ANR Seq-N-Vaq, Charles Hood Foundation, Hearst Foundation, and Groupe Pasteur Mutualité.
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Escherichia coli , Meningitis Bacterianas , Animales , Ratones , Escherichia coli/genética , Anticuerpos Antibacterianos , Bacterias/genética , Inmunoterapia , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Since the beginning of the Coronavirus disease (COVID)-19 pandemic in December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for more than 600 million infections and 6.5 million deaths worldwide. Given the persistence of SARS-CoV-2 and its ability to develop new variants, the implementation of an effective and long-term herd immunity appears to be crucial to overcome the pandemic. While a vast field of research has focused on the role of humoral immunity against SARS-CoV-2, a growing body of evidence suggest that antibodies alone only confer a partial protection against infection of reinfection which could be of high importance regarding the strategic development goals (SDG) of the United Nations (UN) and in particular UN SDG3 that aims towards the realization of good health and well being on a global scale in the context of the COVID-19 pandemic.In this review, we highlight the role of humoral immunity in the host defense against SARS-CoV-2, with a focus on highly neutralizing antibodies. We summarize the results of the main clinical trials leading to an overall disappointing efficacy of convalescent plasma therapy, variable results of monoclonal neutralizing antibodies in patients with COVID-19 but outstanding results for the mRNA based vaccines against SARS-CoV-2. Finally, we advocate that beyond antibody responses, the development of a robust cellular immunity against SARS-CoV-2 after infection or vaccination is of utmost importance for promoting immune memory and limiting disease severity, especially in case of (re)-infection by variant viruses.
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COVID-19 , SARS-CoV-2 , Humanos , Vacunas contra la COVID-19 , Pandemias/prevención & control , Sueroterapia para COVID-19 , Anticuerpos Neutralizantes/uso terapéuticoRESUMEN
Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections.
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Adhesión Bacteriana , Proteínas Bacterianas/fisiología , Fimbrias Bacterianas/fisiología , Neisseria meningitidis/fisiología , Animales , Anticuerpos/uso terapéutico , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Línea Celular , Evaluación Preclínica de Medicamentos , Femenino , Fimbrias Bacterianas/química , Fimbrias Bacterianas/ultraestructura , Humanos , Infecciones Meningocócicas/tratamiento farmacológico , Ratones SCIDRESUMEN
Neisseria meningitidis is the causative agent of cerebrospinal meningitis and that of a rapidly progressing fatal septic shock known as purpura fulminans. Meningococcemia is characterized by bacterial adhesion to human endothelial cells of the microvessels. Host specificity has hampered studies on the role of blood vessels colonization in N. meningitidis associated pathogenesis. In this work, using a humanized model of SCID mice allowing the study of bacterial adhesion to human cells in an in vivo context we demonstrate that meningococcal colonization of human blood vessels is a prerequisite to the establishment of sepsis and lethality. To identify the molecular pathways involved in bacterial virulence, we performed transposon insertion site sequencing (Tn-seq) in vivo. Our results demonstrate that 36% of the genes that are important for growth in the blood of mice are dispensable when bacteria colonize human blood vessels, suggesting that human endothelial cells lining the blood vessels are feeding niches for N. meningitidis in vivo. Altogether, our work proposes a new paradigm for meningococcal virulence in which colonization of blood vessels is associated with metabolic adaptation and sustained bacteremia responsible for sepsis and subsequent lethality.
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Bacteriemia/microbiología , Infecciones Meningocócicas/sangre , Infecciones Meningocócicas/microbiología , Microvasos/microbiología , Neisseria meningitidis/fisiología , Animales , Bacteriemia/sangre , Adhesión Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Neisseria meningitidis/genéticaRESUMEN
UNLABELLED: Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia, affecting infants and adults worldwide. N. meningitidis is also a common inhabitant of the human nasopharynx and, as such, is highly adapted to its niche. During bacteremia, N. meningitidis gains access to the blood compartment, where it adheres to endothelial cells of blood vessels and causes dramatic vascular damage. Colonization of the nasopharyngeal niche and communication with the different human cell types is a major issue of the N. meningitidis life cycle that is poorly understood. Here, highly saturated random transposon insertion libraries of N. meningitidis were engineered, and the fitness of mutations during routine growth and that of colonization of endothelial and epithelial cells in a flow device were assessed in a transposon insertion site sequencing (Tn-seq) analysis. This allowed the identification of genes essential for bacterial growth and genes specifically required for host cell colonization. In addition, after having identified the small noncoding RNAs (sRNAs) located in intergenic regions, the phenotypes associated with mutations in those sRNAs were defined. A total of 383 genes and 8 intergenic regions containing sRNA candidates were identified to be essential for growth, while 288 genes and 33 intergenic regions containing sRNA candidates were found to be specifically required for host cell colonization. IMPORTANCE: Meningococcal meningitis is a common cause of meningitis in infants and adults. Neisseria meningitidis (meningococcus) is also a commensal bacterium of the nasopharynx and is carried by 3 to 30% of healthy humans. Under some unknown circumstances, N. meningitidis is able to invade the bloodstream and cause either meningitis or a fatal septicemia known as purpura fulminans. The onset of symptoms is sudden, and death can follow within hours. Although many meningococcal virulence factors have been identified, the mechanisms that allow the bacterium to switch from the commensal to pathogen state remain unknown. Therefore, we used a Tn-seq strategy coupled to high-throughput DNA sequencing technologies to find genes for proteins used by N. meningitidis to specifically colonize epithelial cells and primary brain endothelial cells. We identified 383 genes and 8 intergenic regions containing sRNAs essential for growth and 288 genes and 33 intergenic regions containing sRNAs required specifically for host cell colonization.
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Endocitosis , Células Endoteliales/microbiología , Células Epiteliales/microbiología , Neisseria meningitidis/genética , Neisseria meningitidis/patogenicidad , ARN Pequeño no Traducido/genética , Factores de Virulencia/genética , Línea Celular , Elementos Transponibles de ADN , Técnicas de Inactivación de Genes , Biblioteca de Genes , Humanos , Mutagénesis Insercional , Neisseria meningitidis/crecimiento & desarrolloRESUMEN
The mechanism by which Neisseria meningitidis becomes invasive is not well understood. Comparative genomics identified the presence of an 8 kb island in strains belonging to invasive clonal complexes. This island was designated MDA for meningococcal disease associated. MDA is highly conserved among meningococcal isolates and its analysis revealed a genomic organization similar to that of a filamentous prophage such as CTXΦ of Vibrio cholerae. Subsequent molecular investigations showed that the MDA island has indeed the characteristics of a filamentous prophage, which can enter into a productive cycle and is secreted using the type IV pilus (tfp) secretin PilQ. At least three genes of the prophage are necessary for the formation of the replicative cytoplasmic form (orf1, orf2 and orf9). Immunolabelling of the phage with antibodies against the major capsid protein, ORF4, confirmed that filamentous particles, about 1200 nm long, covered with ORF4 are present at the bacterial surface forming bundles in some places and interacting with pili. The MDA bacteriophage is able to infect different N. meningitidis strains, using the type IV pili as a receptor via an interaction with the adsorption protein ORF6. Altogether, these data demonstrate that the MDA island encodes a functional prophage able to produce infectious filamentous phage particles.
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Sitios de Ligazón Microbiológica/genética , Inovirus/genética , Neisseria meningitidis/genética , Neisseria meningitidis/virología , Profagos/genética , Receptores Virales/genética , Secuencia de Bases , ADN Viral/genética , Fimbrias Bacterianas/virología , Infecciones Meningocócicas/microbiología , Neisseria meningitidis/patogenicidad , Análisis de Secuencia de ADNRESUMEN
Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.
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Adaptación Fisiológica , Francisella tularensis/fisiología , Isoleucina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Citosol/microbiología , Modelos Animales de Enfermedad , Femenino , Francisella tularensis/genética , Francisella tularensis/crecimiento & desarrollo , Francisella tularensis/metabolismo , Eliminación de Gen , Proteínas de Transporte de Membrana/genética , Ratones Endogámicos BALB C , Fagosomas/microbiología , Tularemia/microbiología , Tularemia/patologíaRESUMEN
Hidradenitis suppurativa (HS) is a skin disease characterized by recurrent nodules or abscesses and chronic suppurating lesions. In the absence of clear pathophysiology, HS is considered to be an inflammatory disease and has no satisfactory medical treatment. Recently, prolonged antimicrobial treatments were shown to improve or resolve HS lesions. We prospectively studied the microbiology of 102 HS lesions sampled from 82 patients using prolonged bacterial cultures and bacterial metagenomics on 6 samples. Staphylococcus lugdunensis was cultured as a unique or predominant isolate from 58% of HS nodules and abscesses, and a polymicrobial anaerobic microflora comprising strict anaerobes, milleri group streptococci, and actinomycetes was found in 24% of abscesses or nodules and in 87% of chronic suppurating lesions. These data show that bacteria known to cause soft tissue and skin infections are associated with HS lesions. Whether these pathogens are the cause of the lesions or are secondary infectious agents, these findings support targeted antimicrobial treatment of HS.
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Hidradenitis Supurativa/epidemiología , Hidradenitis Supurativa/microbiología , Bacterias Anaerobias/clasificación , Bacterias Anaerobias/genética , Bacterias Anaerobias/aislamiento & purificación , Biodiversidad , Francia/epidemiología , Humanos , MetagenómicaRESUMEN
In order to develop a successful infectious cycle, intracellular bacterial pathogens must be able to adapt their metabolism to optimally utilize the nutrients available in the cellular compartments and tissues where they reside. Francisella tularensis, the agent of the zoonotic disease tularaemia, is a highly infectious bacterium for a large number of animal species. This bacterium replicates in its mammalian hosts mainly in the cytosol of infected macrophages. We report here the identification of a novel amino acid transporter of the major facilitator superfamily of secondary transporters that is required for bacterial intracellular multiplication and systemic dissemination. We show that inactivation of this transporter does not affect phagosomal escape but prevents multiplication in the cytosol of all cell types tested. Remarkably, the intracellular growth defect of the mutant was fully and specifically reversed by addition of asparagine or asparagine-containing dipeptides as well as by simultaneous addition of aspartic acid and ammonium. Importantly, bacterial virulence was also restored in vivo, in the mouse model, by asparagine supplementation. This work unravels thus, for the first time, the importance of asparagine for cytosolicmultiplication of Francisella. Amino acid transporters are likely to constitute underappreciated players in bacterial intracellular parasitism.
Asunto(s)
Sistemas de Transporte de Aminoácidos/genética , Asparagina/metabolismo , Proteínas Bacterianas/genética , Francisella tularensis/crecimiento & desarrollo , Compuestos de Amonio/farmacología , Animales , Asparagina/farmacología , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Proteínas Bacterianas/farmacocinética , Línea Celular Tumoral , Francisella tularensis/metabolismo , Francisella tularensis/patogenicidad , Células Hep G2 , Humanos , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Fagosomas/microbiología , Tularemia/microbiologíaRESUMEN
Francisella tularensis is a highly virulent bacterium responsible for the zoonotic disease tularemia. It is a facultative intracellular pathogen that replicates in the cytoplasm of host cells, particularly in macrophages. Here we show that F. tularensis live vaccine strain (LVS) expresses a novel small RNA (sRNA), which modulates the virulence capacities of the bacterium. When this sRNA, designated FtrC (for Francisella tularensis RNA C), is expressed at high levels, F. tularensis replicates in macrophages less efficiently than the wild-type parent strain. Similarly, high expression of FtrC reduces the number of viable bacteria recovered from the spleen and liver of infected mice. Our data demonstrate that expression of gene FTL_1293 is regulated by FtrC. Furthermore, we show by in vitro gel shift assays that FtrC interacts specifically with FTL_1293 mRNA and that this happens independently of the RNA chaperone Hfq. Remarkably, FtrC interacts only with full-length FTL_1293 mRNA. These results, combined with a bioinformatic analysis, indicate that FtrC interacts with the central region of the mRNA and hence does not act by sterically hindering access of the ribosome to the mRNA. We further show that gene FTL_1293 is not required for F. tularensis virulence in vitro or in vivo, which indicates that another unidentified FtrC target modulates the virulence capacity of the bacterium.
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Francisella tularensis/genética , Francisella tularensis/patogenicidad , ARN Bacteriano/genética , ARN no Traducido/genética , Animales , Secuencia de Bases , Femenino , Regulación Bacteriana de la Expresión Génica/genética , Espacio Intracelular/microbiología , Macrófagos/citología , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Especificidad de la EspecieRESUMEN
The ability of Neisseria meningitidis to establish efficient interaction with host cells is crucial for its survival. We recently demonstrated that an entire operon containing genes NMA1802 to NMA1806 was overexpressed during the early stage of the colonization process. In this work, we investigated whether upregulation of the expression of this operon facilitated the ability of N. meningitidis to adapt to growth on host cells. Using a strain displaying an inducible operon, we demonstrated that the NMA1802-NMA1806 cell-contact-regulated operon could potentially improve the adaptability of meningococcus during growth on the cell surface through enhanced generation of variants.
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Adhesión Bacteriana , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Neisseria meningitidis/fisiología , Operón , Perfilación de la Expresión Génica , Humanos , Neisseria meningitidis/genéticaRESUMEN
A scientist in our laboratory was accidentally infected while working with Z5463, a Neisseria meningitidis serogroup A strain. She developed severe symptoms (fever, meningism, purpuric lesions) that fortunately evolved with antibiotic treatment to complete recovery. Pulse-field gel electrophoresis confirmed that the isolate obtained from the blood culture (Z5463BC) was identical to Z5463, more precisely to a fourth subculture of this strain used the week before the contamination (Z5463PI). In order to get some insights into genomic modifications that can occur in vivo, we sequenced these three isolates. All the strains contained a mutated mutS allele and therefore displayed an hypermutator phenotype, consistent with the high number of mutations (SNP, Single Nucleotide Polymorphism) detected in the three strains. By comparing the number of SNP in all three isolates and knowing the number of passages between Z5463 and Z5463PI, we concluded that around 25 bacterial divisions occurred in the human body. As expected, the in vivo passage is responsible for several modifications of phase variable genes. This genomic study has been completed by transcriptomic and phenotypic studies, showing that the blood strain used a different haemoglobin-linked iron receptor (HpuA/B) than the parental strains (HmbR). Different pilin variants were found after the in vivo passage, which expressed different properties of adhesion. Furthermore the deletion of one gene involved in LOS biosynthesis (lgtB) results in Z5463BC expressing a different LOS than the L9 immunotype of Z2491. The in vivo passage, despite the small numbers of divisions, permits the selection of numerous genomic modifications that may account for the high capacity of the strain to disseminate.
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Variación Antigénica , Infección Hospitalaria/microbiología , Variación Genética , Infecciones Meningocócicas/microbiología , Neisseria meningitidis/genética , Neisseria meningitidis/inmunología , Accidentes de Trabajo , Adulto , Variación Antigénica/genética , Variación Antigénica/fisiología , Infección Hospitalaria/genética , Infección Hospitalaria/inmunología , Femenino , Genotipo , Humanos , Personal de Laboratorio Clínico , Infecciones Meningocócicas/genética , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/transmisión , Neisseria meningitidis/fisiología , FenotipoRESUMEN
Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. This facultative intracellular bacterium replicates in vivo mainly inside macrophages and therefore has developed strategies to resist this stressful environment. Here, we identified a novel genetic locus that is important for stress resistance and intracellular survival of F. tularensis. In silico and transcriptional analyses suggest that this locus (genes FTL_0200 to FTL_0209 in the live vaccine strain [LVS]) constitutes an operon controlled by the alternative sigma factor σ³². The first gene, FTL_0200, encodes a putative AAA+ ATPase of the MoxR subfamily. Insertion mutagenesis into genes FTL_0200, FTL_0205, and FTL_0206 revealed a role for the locus in both intracellular multiplication and in vivo survival of F. tularensis. Deletion of gene FTL_0200 led to a mutant bacterium with increased vulnerability to various stress conditions, including oxidative and pH stresses. Proteomic analyses revealed a pleiotropic impact of the ΔFTL_0200 deletion, supporting a role as a chaperone for FTL_0200. This is the first report of a role for a MoxR family member in bacterial pathogenesis. This class of proteins is remarkably conserved among pathogenic species and may thus constitute a novel player in bacterial virulence.
Asunto(s)
Francisella tularensis/genética , Francisella tularensis/patogenicidad , Genes Bacterianos/genética , Chaperonas Moleculares/genética , Estrés Fisiológico/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Southern Blotting , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tularemia/genética , Tularemia/metabolismo , Virulencia/genéticaRESUMEN
BACKGROUND: Regulation of bacterial gene expression by small RNAs (sRNAs) have proved to be important for many biological processes. Francisella tularensis is a highly pathogenic Gram-negative bacterium that causes the disease tularaemia in humans and animals. Relatively little is known about the regulatory networks existing in this organism that allows it to survive in a wide array of environments and no sRNA regulators have been identified so far. RESULTS: We have used a combination of experimental assays and in silico prediction to identify sRNAs in F. tularensis strain LVS. Using a cDNA cloning and sequencing approach we have shown that F. tularensis expresses homologues of several sRNAs that are well-conserved among diverse bacteria. We have also discovered two abundant putative sRNAs that share no sequence similarity or conserved genomic context with any previously annotated regulatory transcripts. Deletion of either of these two loci led to significant changes in the expression of several mRNAs that likely include the cognate target(s) of these sRNAs. Deletion of these sRNAs did not, however, significantly alter F. tularensis growth under various stress conditions in vitro, its replication in murine cells, or its ability to induce disease in a mouse model of F. tularensis infection. We also conducted a genome-wide in silico search for intergenic loci that suggests F. tularensis encodes several other sRNAs in addition to the sRNAs found in our experimental screen. CONCLUSION: Our findings suggest that F. tularensis encodes a significant number of non-coding regulatory RNAs, including members of well conserved families of structural and housekeeping RNAs and other poorly conserved transcripts that may have evolved more recently to help F. tularensis deal with the unique and diverse set of environments with which it must contend.
Asunto(s)
Francisella tularensis/genética , ARN Bacteriano/análisis , ARN Bacteriano/genética , Animales , Vacunas Bacterianas/inmunología , Secuencia de Bases , Northern Blotting , Clonación Molecular , Biología Computacional , ADN Complementario/genética , Francisella tularensis/inmunología , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Espacio Intracelular/microbiología , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Transporte de ARN/genética , ARN Bacteriano/química , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas Atenuadas/inmunologíaRESUMEN
Mycobacterial identification is based on several methods: conventional biochemical tests that require several weeks for accurate identification, and molecular tools that are now routinely used. However, these techniques are expensive and time-consuming. In this study, an alternative method was developed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This approach allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycobacterial cells. We engineered a strategy based on specific profiles in order to identify the most clinically relevant species of mycobacteria. To validate the mycobacterial database, a total of 311 strains belonging to 31 distinct species and 4 species complexes grown in Löwenstein-Jensen (LJ) and liquid (mycobacterium growth indicator tube [MGIT]) media were analyzed. No extraction step was required. Correct identifications were obtained for 97% of strains from LJ and 77% from MGIT media. No misidentification was noted. Our results, based on a very simple protocol, suggest that this system may represent a serious alternative for clinical laboratories to identify mycobacterial species.
Asunto(s)
Técnicas Bacteriológicas/métodos , Mycobacterium/química , Mycobacterium/clasificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tuberculosis/diagnóstico , Tuberculosis/microbiología , Humanos , Mycobacterium/crecimiento & desarrolloRESUMEN
In order to adapt to changing environments, bacteria have evolved two-component systems (TCSs) that are able to sense and respond to environmental stimuli. The signal perception relies on a sensor protein whose activation allows rapid adaptation through transcriptional regulation achieved by the regulatory protein. The ability to adhere to and grow on the surface of human host cells is an absolute requirement for many pathogens, including Neisseria meningitidis, in order to colonize new hosts and to disseminate inside their host. Among the four TCSs encoded in the meningococcus genome, only the PhoQ (MisS)/PhoP (MisR) system has been shown to constitute a functional signal transduction circuit. To investigate the involvement of this TCS in the adaptation process requisite for host cell colonization, we have tested the ability to grow on host cells of a mutant inactivated for the sensor of the TCS. Our results demonstrate the involvement of the TCS in the adaptation of the meningococcus to growth on host cells. We show that the expression of the PhoQ (MisS)/PhoP (MisR) TCS is cell-contact controlled. Furthermore, this TCS controls the regulation of a group of genes, the REP2 regulon, previously shown to be cell-contact regulated and to encode functions crucial for the adaptation of the bacterium to host cell colonization. Thus, we provide evidence that one of the four TCSs existing in N. meningitidis contributes to the adaptation of the pathogen to growth on host cells.