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1.
Artículo en Inglés | MEDLINE | ID: mdl-33852710

RESUMEN

Despite the widespread use of chlorhexidine (CHX) to prevent infection, data regarding the in vitro action of CHX against methicillin-resistant Staphylococcus aureus (MRSA) are limited. Clinical isolates from Hospital das Clinicas, Sao Paulo, Brazil, identified during 2002/2003 and 2012/2013 were studied to describe the susceptibility to CHX and mupirocin, molecular characteristics, and virulence profile of MRSA. Susceptibility test to Mupirocin was performed by the disk diffusion method and to CHX by the agar dilution technique. PCR for virulence genes, mecA gene and Staphylococcal Cassette Chromosome mec (SCCmec) types were investigated as well. Mupirocin- and CHX-resistant isolates were sequenced using the IlluminaTM plataform. Two hundred and sixteen MRSA clinical isolates were evaluated: 154 from infected and 62 from colonized patients. Resistance to mupirocin was observed in four isolates assigned as SCCmec type III and STs (ST05; ST239 and ST105) carrying mupA and blaZ, two of them co-harboring the ileS gene. Only one isolate assigned as SCCmec type III was resistant to CHX (MIC of 8.0 µg.mL-1) and harbored the qacA gene. Resistance to chlorhexidine and mupirocin were found in isolates carrying qacA and mupA in our hospital. Since these genes are plasmid-mediated, this finding draws attention to the potential spread of resistance to mupirocin in our hospital.


Asunto(s)
Antibacterianos/farmacología , Clorhexidina/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Mupirocina/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Bacterianas/genética , Brasil , Niño , Preescolar , ADN Bacteriano/genética , Femenino , Hospitales de Enseñanza , Humanos , Lactante , Masculino , Staphylococcus aureus Resistente a Meticilina/genética , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Análisis de Secuencia de ADN , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Virulencia , Adulto Joven
2.
J Med Microbiol ; 70(4)2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33830906

RESUMEN

Introduction. Nitrofurantoin is one of the preferred antibiotics in the treatment of uropathogenic multidrug-resistant (MDR) infections. However, resistance to nitrofurantoin in extensively drug-resistant (XDR) bacteria has severely limited the treatment options.Gap statement. Information related to co-resistance or collateral sensitivity (CS) with reference to nitrofurantoin resistant bacteria is limited.Aim. To study the potential of nitrofurantoin resistance as an indicator of the XDR phenotype in Enterobacteriaceae.Methods. One hundred (45 nitrofurantoin-resistant, 21 intermediately resistant and 34 nitrofurantoin-susceptible) Enterobacteriaceae were analysed in this study. Antibiotic susceptibility testing (AST) against nitrofurantoin and 17 other antimicrobial agents across eight different classes was performed by using the Vitek 2.0 system. The isolates were screened for the prevalence of acquired antimicrobial resistance (AMR) and efflux pump genes by PCR.Results. In total, 51 % of nitrofurantoin-resistant and 28 % of intermediately nitrofurantoin resistant isolates exhibited XDR characteristics, while only 3 % of nitrofurantoin-sensitive isolates were XDR (P=0.0001). Significant co-resistance was observed between nitrofurantoin and other tested antibiotics (ß-lactam, cephalosporin, carbapenem, aminoglycoside and tetracycline). Further, the prevalence of AMR and efflux pump genes was higher in the nitrofurantoin-resistant strains compared to the susceptible isolates. A strong association was observed between nitrofurantoin resistance and the presence of bla PER-1, bla NDM-1, bla OXA-48, ant(2) and oqxA-oqxB genes. Tigecycline (84 %) and colistin (95 %) were the only antibiotics to which the majority of the isolates were susceptible.Conclusion. Nitrofurantoin resistance could be an indicator of the XDR phenotype among Enterobacteriaceae, harbouring multiple AMR and efflux pump genes. Tigecycline and colistin are the only antibiotics that could be used in the treatment of such XDR infections. A deeper understanding of the co-resistance mechanisms in XDR pathogens and prescription of AST-based appropriate combination therapy may help mitigate this problem.


Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Enterobacteriaceae/efectos de los fármacos , Nitrofurantoína/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/fisiología , Infecciones por Enterobacteriaceae/microbiología , Humanos , Pruebas de Sensibilidad Microbiana
3.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33809708

RESUMEN

A typical feature of proteins from the rhodopsin family is the sensitivity of their absorption band maximum to protein amino acid composition. For this reason, studies of these proteins often require methodologies that determine spectral shift caused by amino acid substitutions. Generally, quantum mechanics/molecular mechanics models allow for the calculation of a substitution-induced spectral shift with high accuracy, but their application is not always easy and requires special knowledge. In the present study, we propose simple models that allow us to estimate the direct effect of a charged or polar residue substitution without extensive calculations using only rhodopsin three-dimensional structure and plots or tables that are provided in this article. The models are based on absorption maximum values calculated at the SORCI+Q level of theory for cis- and trans-forms of retinal protonated Schiff base in an external electrostatic field of charges and dipoles. Each value corresponds to a certain position of a charged or polar residue relative to the retinal chromophore. The proposed approach was evaluated against an example set consisting of twelve bovine rhodopsin and sodium pumping rhodopsin mutants. The limits of the applicability of the models are also discussed. The results of our study can be useful for the interpretation of experimental data and for the rational design of rhodopsins with required spectral properties.


Asunto(s)
Aminoácidos/química , Proteínas Bacterianas/química , Modelos Moleculares , Rodopsina/química , Análisis Espectral , Electricidad Estática , Sustitución de Aminoácidos , Animales , Bovinos , Mutación/genética , Protones , Rodopsina/genética , Bases de Schiff/química
4.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33809732

RESUMEN

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.


Asunto(s)
Paenibacillus polymyxa/metabolismo , Riboswitch/genética , Serina/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Codón/genética , Secuencia Conservada , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Nitrogenasa/metabolismo , Conformación de Ácido Nucleico , Motivos de Nucleótidos/genética , Paenibacillus polymyxa/efectos de los fármacos , Paenibacillus polymyxa/genética , Paenibacillus polymyxa/crecimiento & desarrollo , ARN Bacteriano/química , ARN Bacteriano/genética , Serina/farmacología
5.
Biomolecules ; 11(3)2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33810129

RESUMEN

Global processes, such as climate change, frequent and distant travelling and population growth, increase the risk of viral infection spread. Unfortunately, the number of effective and accessible medicines for the prevention and treatment of these infections is limited. Therefore, in recent years, efforts have been intensified to develop new antiviral medicines or vaccines. In this review article, the structure and activity of the most promising antiviral cyanobacterial products are presented. The antiviral cyanometabolites are mainly active against the human immunodeficiency virus (HIV) and other enveloped viruses such as herpes simplex virus (HSV), Ebola or the influenza viruses. The majority of the metabolites are classified as lectins, monomeric or dimeric proteins with unique amino acid sequences. They all show activity at the nanomolar range but differ in carbohydrate specificity and recognize a different epitope on high mannose oligosaccharides. The cyanobacterial lectins include cyanovirin-N (CV-N), scytovirin (SVN), microvirin (MVN), Microcystisviridis lectin (MVL), and Oscillatoria agardhii agglutinin (OAA). Cyanobacterial polysaccharides, peptides, and other metabolites also have potential to be used as antiviral drugs. The sulfated polysaccharide, calcium spirulan (CA-SP), inhibited infection by enveloped viruses, stimulated the immune system's response, and showed antitumor activity. Microginins, the linear peptides, inhibit angiotensin-converting enzyme (ACE), therefore, their use in the treatment of COVID-19 patients with injury of the ACE2 expressing organs is considered. In addition, many cyanobacterial extracts were revealed to have antiviral activities, but the active agents have not been identified. This fact provides a good basis for further studies on the therapeutic potential of these microorganisms.


Asunto(s)
Antivirales/química , Cianobacterias/química , VIH/efectos de los fármacos , Lectinas/farmacología , Polisacáridos/farmacología , Simplexvirus/efectos de los fármacos , Fármacos Anti-VIH/farmacología , Antineoplásicos/farmacología , Antivirales/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Carbohidratos/química , Carbohidratos/farmacología , Cianobacterias/metabolismo , Infecciones por VIH/tratamiento farmacológico , Humanos , Lectinas/química , Lectinas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo
6.
Zhonghua Yu Fang Yi Xue Za Zhi ; 55(4): 506-511, 2021 Apr 06.
Artículo en Chino | MEDLINE | ID: mdl-33858063

RESUMEN

Objective: To explore the clinical application value of modified carbapenem inactivation test (mCIM) combined with EDTA-modified carbapenem inactivation test (eCIM) for detecting the carbapenemase of CRE isolated from infected patients in clinical diagnosis and infection control of CRE infection. Methods: Drug resistance of seventy eight non-repetitive enterobacteriaceae resistant to carbapenem antibiotics, which were isolated from clinically infected patients from January 2017 to December 2017 in the Tianjin Medical University General Hospital, was retrospectively analyzed. Meanwhile, Vitek2 Compact automatic bacterial identification instrument was used to identify the species and detect its minimum inhibitory concentration (MIC) for ertapenem and imipenem. Carbapenemase genes blaKPC, blaNDM, blaIMP and blaVIM were detected by PCR test, the genotype was determined by gene sequencing as the gold standard, and mCIM Combined eCIM test was used for carbapenemase detection of collected bacteria. Using PCR results as the standard, the sensitivity, specificity, positive predictive value, negative predictive value and Cohen' Kappa value of mCIM and eCIM were calculated, and the consistency of the combined detection results of mCIM and eCIM and PCR results was checked. Results: Seventy eight carbapenem-resistant Enterobacteriaceae were detected by PCR, 74 carbapenemase gene positive and 4 negative, including 60 blaKPC-2 positive, 2 blaNDM-1 positive and blaNDM-5 positive Of the 7 strains, 3 strains were positive for blaNDM-9, 1 strain was positive for blaIMP-4, and 1 strain was both positive for blaKPC-2 and blaNDM-1. The sensitivity of mCIM to detect carbapenemase is 100%, the specificity is 100%, and the Kappa value is 1.000; the combined detection of mCIM and eCIM for serine carbapenemase has a sensitivity of 100%, the specificity is 100%, and the Kappa value is 1.000; The sensitivity of combined detection of metallo-ß-lactamase is 92.9%, the specificity is 100%, and the Kappa value is 0.955. Conclusions: The combined test of mCIM and eCIM which is used to detect carbapenemase in CRE costs low, doesn't require special reagents and equipment, has strong practicability, simple operation, and easy interpretation of results. According to the different genotypes of CRE bacteria, it provides important clinical diagnostic evidence for clinical CRE diagnosis, precise antimicrobial treatment and infection control.


Asunto(s)
Enterobacteriaceae Resistentes a los Carbapenémicos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Carbapenémicos/farmacología , Ácido Edético , Humanos , Pruebas de Sensibilidad Microbiana , Estudios Retrospectivos , beta-Lactamasas/genética
7.
Nat Commun ; 12(1): 1986, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790266

RESUMEN

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.


Asunto(s)
Proteínas Bacterianas/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/fisiología , Algoritmos , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Cromatografía Liquida , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Espectrometría de Masas , Liasas de Fósforo-Oxígeno/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Temperatura
8.
Pestic Biochem Physiol ; 174: 104814, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33838714

RESUMEN

Toxin-antitoxin (TA) systems are comprised of a toxin and its antidote antitoxin and are widely present in bacterial and in eukaryotic systems. However, no work regarding TA systems has been reported in insects. We characterized the Kid-Kis and MazF-MazE TA systems in Spodoptera frugiperda cells and Mythimna separata embryos and observed that the Kid and MazF toxins were highly toxic. In Sf9 cells transfected with Kid plasmid and MazF alone, the apoptosis rate was 24.37% and 29.47%, respectively. Whereas the toxicity of their cognate antitoxins were limited. Both apoptosis and necrosis were induced by the two toxins. Both the Kis and MazE antitoxins partly neutralized toxicity in a dose-dependent manner, with MazE accomplishing almost full neutralization at a 1:4 toxin:antitoxin ratio, the cell survival rate was 81% and 97%, respectively. Our results indicate that MazF-MazE is a good candidate module for application in insects, such as in developing new sterile insect technique (SIT).


Asunto(s)
Toxinas Bacterianas , Proteínas de Escherichia coli , Animales , Proteínas Bacterianas , Proteínas de Unión al ADN , Células Sf9
9.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-33802668

RESUMEN

Avibactam belongs to the new class of diazabicyclooctane ß-lactamase inhibitors. Its inhibitory spectrum includes class A, C and D enzymes, including P. aeruginosa AmpC. Nonetheless, recent reports have revealed strain-dependent avibactam AmpC induction. In the present work, we wanted to assess the mechanistic basis underlying AmpC induction and determine if derepressed PDC-X mutated enzymes from ceftazidime/avibactam-resistant clinical isolates were further inducible. We determined avibactam concentrations that half-maximally inhibited (IC50) bocillin FL binding. Inducer ß-lactams were also studied as comparators. Live cells' time-course penicillin-binding proteins (PBPs) occupancy of avibactam was studied. To assess the ampC induction capacity of avibactam and comparators, qRT-PCR was performed in wild-type PAO1, PBP4, triple PBP4, 5/6 and 7 knockout derivatives and two ceftazidime/avibactam-susceptible/resistant XDR clinical isolates belonging to the epidemic high-risk clone ST175. PBP4 inhibition was observed for avibactam and ß-lactam comparators. Induction capacity was consistently correlated with PBP4 binding affinity. Outer membrane permeability-limited PBP4 binding was observed in the live cells' assay. As expected, imipenem and cefoxitin showed strong induction in PAO1, especially for carbapenem; avibactam induction was conversely weaker. Overall, the inducer effect was less remarkable in ampC-derepressed mutants and nonetheless absent upon avibactam exposure in the clinical isolates harboring mutated AmpC variants and their parental strains.


Asunto(s)
Compuestos de Azabiciclo/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Mutación/genética , Proteínas de Unión a las Penicilinas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , beta-Lactamasas/metabolismo , Proteínas Bacterianas/metabolismo , Cefoxitina/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Humanos , Imipenem/farmacología , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos
10.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-33805767

RESUMEN

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell's cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.


Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Infección Hospitalaria/tratamiento farmacológico , Enterocolitis Seudomembranosa/tratamiento farmacológico , Enterotoxinas/antagonistas & inhibidores , ADP-Ribosilación/efectos de los fármacos , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/deficiencia , Actinas/genética , Antibacterianos/uso terapéutico , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sitios de Unión , /genética , Infección Hospitalaria/metabolismo , Infección Hospitalaria/microbiología , Infección Hospitalaria/patología , Endocitosis/efectos de los fármacos , Enterocolitis Seudomembranosa/metabolismo , Enterocolitis Seudomembranosa/microbiología , Enterocolitis Seudomembranosa/patología , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína
11.
Nat Commun ; 12(1): 2333, 2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33879791

RESUMEN

Acaryochloris marina is one of the cyanobacterial species that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of A. marina photosystem I (PSI) reaction center, determined by cryo-electron microscopy at 2.58 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments distinct from other type I reaction centers. The paired chlorophyll, or special pair (also referred to as P740 in this case), is a dimer of chlorophyll d and its epimer chlorophyll d'. The primary electron acceptor is pheophytin a, a metal-less chlorin. We show the architecture of this PSI reaction center is composed of 11 subunits and we identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis.


Asunto(s)
Proteínas Bacterianas/química , Cianobacterias/química , Complejo de Proteína del Fotosistema I/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clorofila/química , Clorofila/metabolismo , Microscopía por Crioelectrón , Cianobacterias/genética , Cianobacterias/metabolismo , Transporte de Electrón , Luz , Modelos Moleculares , Oxígeno/metabolismo , Fotosíntesis , Complejo de Proteína del Fotosistema I/genética , Complejo de Proteína del Fotosistema I/metabolismo , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Electricidad Estática
12.
J Dent Res ; 100(5): 542-548, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33876976

RESUMEN

Streptococcus sobrinus is an etiologic cause of dental caries (tooth decay) in humans. Our knowledge of S. sobrinus is scant despite the organism's important role in oral health. It is widely believed that S. sobrinus lacks the natural competence pathways that are used by other streptococci to regulate growth, virulence, and quorum sensing. The lack of natural competence has also prevented genetic manipulation of S. sobrinus, limiting our knowledge of its pathogenicity. We discovered that most strains of S. sobrinus contain a new class of the ComRS competence system. Although S. sobrinus is typically placed among the mutans group streptococci, the S. sobrinus ComRS system is most similar to the competence pathways in the salivarius group. Unlike all other ComRS systems, the S. sobrinus pathway contains 2 copies of the transcriptional regulator ComR and has a peptide pheromone (XIP) that lacks any aromatic amino acids. Synthetic XIP enables transformation of S. sobrinus with plasmid or linear DNA, and we leverage this newfound genetic tractability to confirm that only 1 of the ComR homologs is required for induced competence while the other appears to suppress competence. Exogenous XIP increases the expression of bacteriocin gene clusters and produces an antimicrobial response that inhibits growth of S. mutans. We also identified 2 strains of S. sobrinus that appear to be "cheaters" by either not responding to or not producing XIP. We show how a recombination event in the nonresponsive strain could restore function of the ComRS pathway but delete the gene encoding XIP. Thus, the S. sobrinus ComRS pathway provides new tools for studying this pathogen and offers a lens into the evolution of ecological cheaters.


Asunto(s)
Caries Dental , Streptococcus sobrinus , Proteínas Bacterianas/genética , Humanos , Percepción de Quorum , Streptococcus , Streptococcus mutans/genética , Streptococcus sobrinus/genética
13.
mBio ; 12(2)2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33879592

RESUMEN

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to gamma interferon (IFN-γ) or nutrient/oxygen deprivation of in vitro-infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analyzed their corresponding CD4 T cell phenotype and vaccine protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination, and against the overexpressing strain, vaccination with MPT70 conferred protection similar to vaccination with ESAT-6. Together, our data indicate that high in vivo antigen expression drives T cells toward terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune balance in favor of the host.IMPORTANCE Tuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current coronavirus disease 2019 (COVID-19) pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunization with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.


Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/farmacología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/prevención & control , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/microbiología , Diferenciación Celular/inmunología , Modelos Animales de Enfermedad , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Expresión Génica , Genes Bacterianos , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/microbiología
14.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33799806

RESUMEN

Carbonic anhydrases (CAs) have been identified as ideal catalysts for CO2 sequestration. Here, we report the sequence and structural analyses as well as the molecular dynamics (MD) simulations of four γ-CAs from thermophilic bacteria. Three of these, Persephonella marina, Persephonella hydrogeniphila, and Thermosulfidibacter takaii originate from hydrothermal vents and one, Thermus thermophilus HB8, from hot springs. Protein sequences were retrieved and aligned with previously characterized γ-CAs, revealing differences in the catalytic pocket residues. Further analysis of the structures following homology modeling revealed a hydrophobic patch in the catalytic pocket, presumed important for CO2 binding. Monitoring of proton shuttling residue His69 (P. marina γ-CA numbering) during MD simulations of P. hydrogeniphila and P. marina's γ-CAs (γ-PhCA and γ-PmCA), showed a different behavior to that observed in the γ-CA of Escherichia coli, which periodically coordinates Zn2+. This work also involved the search for hotspot residues that contribute to interface stability. Some of these residues were further identified as key in protein communication via betweenness centrality metric of dynamic residue network analysis. T. takaii's γ-CA showed marginally lower thermostability compared to the other three γ-CA proteins with an increase in conformations visited at high temperatures being observed. Hydrogen bond analysis revealed important interactions, some unique and others common in all γ-CAs, which contribute to interface formation and thermostability. The seemingly thermostable γ-CA from T. thermophilus strangely showed increased unsynchronized residue motions at 423 K. γ-PhCA and γ-PmCA were, however, preliminarily considered suitable as prospective thermostable CO2 sequestration agents.


Asunto(s)
Proteínas Bacterianas/metabolismo , Biomineralización , Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/metabolismo , Secuencia de Aminoácidos , Bacterias/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dióxido de Carbono/química , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/genética , Catálisis , Dominio Catalítico , Simulación por Computador , Manantiales de Aguas Termales/microbiología , Respiraderos Hidrotermales/microbiología , Simulación de Dinámica Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Temperatura , Thermus thermophilus/enzimología
15.
Nat Commun ; 12(1): 2085, 2021 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-33837194

RESUMEN

Long-term infection of the stomach with Helicobacter pylori can cause gastric cancer. However, the mechanisms by which the bacteria adapt to the stomach environment are poorly understood. Here, we show that a small non-coding RNA of H. pylori (HPnc4160, also known as IsoB or NikS) regulates the pathogen's adaptation to the host environment as well as bacterial oncoprotein production. In a rodent model of H. pylori infection, the genomes of bacteria isolated from the stomach possess an increased number of T-repeats upstream of the HPnc4160-coding region, and this leads to reduced HPnc4160 expression. We use RNA-seq and iTRAQ analyses to identify eight targets of HPnc4160, including genes encoding outer membrane proteins and oncoprotein CagA. Mutant strains with HPnc4160 deficiency display increased colonization ability of the mouse stomach, in comparison with the wild-type strain. Furthermore, HPnc4160 expression is lower in clinical isolates from gastric cancer patients than in isolates derived from non-cancer patients, while the expression of HPnc4160's targets is higher in the isolates from gastric cancer patients. Therefore, the small RNA HPnc4160 regulates H. pylori adaptation to the host environment and, potentially, gastric carcinogenesis.


Asunto(s)
Adaptación Fisiológica/genética , Infecciones por Helicobacter/patología , Helicobacter pylori/fisiología , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Neoplasias Gástricas/microbiología , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Carcinogénesis , Modelos Animales de Enfermedad , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Regulación Bacteriana de la Expresión Génica/fisiología , Genoma Bacteriano/genética , Gerbillinae , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Helicobacter pylori/patogenicidad , Interacciones Microbiota-Huesped , Humanos , Masculino , Mutación , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , RNA-Seq , Neoplasias Gástricas/patología
16.
Nat Commun ; 12(1): 2261, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33859194

RESUMEN

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.


Asunto(s)
Lignina/metabolismo , Ingeniería Metabólica/métodos , Pseudomonas putida/metabolismo , Succinatos/metabolismo , Álcalis/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Basidiomycota/enzimología , Basidiomycota/genética , Técnicas Biosensibles , Burkholderia/enzimología , Burkholderia/genética , Carbono/metabolismo , Ciclo del Ácido Cítrico/genética , Ácidos Cumáricos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microbiología Industrial/métodos , Lignina/química , Prueba de Estudio Conceptual , Pseudomonas putida/genética
17.
Nat Commun ; 12(1): 1396, 2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33654096

RESUMEN

Increasing numbers of protein interactions have been identified in high-throughput experiments, but only a small proportion have solved structures. Recently, sequence coevolution-based approaches have led to a breakthrough in predicting monomer protein structures and protein interaction interfaces. Here, we address the challenges of large-scale interaction prediction at residue resolution with a fast alignment concatenation method and a probabilistic score for the interaction of residues. Importantly, this method (EVcomplex2) is able to assess the likelihood of a protein interaction, as we show here applied to large-scale experimental datasets where the pairwise interactions are unknown. We predict 504 interactions de novo in the E. coli membrane proteome, including 243 that are newly discovered. While EVcomplex2 does not require available structures, coevolving residue pairs can be used to produce structural models of protein interactions, as done here for membrane complexes including the Flagellar Hook-Filament Junction and the Tol/Pal complex.


Asunto(s)
Aminoácidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Evolución Molecular , Genoma Bacteriano , Mapeo de Interacción de Proteínas , Proteínas Bacterianas/química , Secuencia de Bases , Escherichia coli/genética , Células Eucariotas/metabolismo , Proteínas de la Membrana/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Proteoma/metabolismo
18.
Nat Commun ; 12(1): 1422, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658492

RESUMEN

Trans-acyltransferase polyketide synthases (trans-AT PKSs) are bacterial multimodular enzymes that biosynthesize diverse pharmaceutically and ecologically important polyketides. A notable feature of this natural product class is the existence of chemical hybrids that combine core moieties from different polyketide structures. To understand the prevalence, biosynthetic basis, and evolutionary patterns of this phenomenon, we developed transPACT, a phylogenomic algorithm to automate global classification of trans-AT PKS modules across bacteria and applied it to 1782 trans-AT PKS gene clusters. These analyses reveal widespread exchange patterns suggesting recombination of extended PKS module series as an important mechanism for metabolic diversification in this natural product class. For three plant-associated bacteria, i.e., the root colonizer Gynuella sunshinyii and the pathogens Xanthomonas cannabis and Pseudomonas syringae, we demonstrate the utility of this computational approach for uncovering cryptic relationships between polyketides, accelerating polyketide mining from fragmented genome sequences, and discovering polyketide variants with conserved moieties of interest. As natural combinatorial hybrids are rare among the more commonly studied cis-AT PKSs, this study paves the way towards evolutionarily informed, rational PKS engineering to produce chimeric trans-AT PKS-derived polyketides.


Asunto(s)
Aciltransferasas/genética , Proteínas Bacterianas/genética , Filogenia , Sintasas Poliquetidas/genética , Policétidos/metabolismo , Aciltransferasas/metabolismo , Algoritmos , Arabidopsis/microbiología , Proteínas Bacterianas/metabolismo , Evolución Molecular , Genoma Bacteriano , Células HeLa , Humanos , Lactonas/metabolismo , Macrólidos/metabolismo , Familia de Multigenes , Piperidonas/química , Plantas/microbiología , Sintasas Poliquetidas/metabolismo , Policétidos/química , Pseudomonas syringae/metabolismo , Xanthomonas/metabolismo , Xanthomonas/patogenicidad
19.
Nat Commun ; 12(1): 1513, 2021 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-33686068

RESUMEN

3-Hydroxypropionic acid (3HP), an important three carbon (C3) chemical, is designated as one of the top platform chemicals with an urgent need for improved industrial production. Halomonas bluephagenesis shows the potential as a chassis for competitive bioproduction of various chemicals due to its ability to grow under an open, unsterile and continuous process. Here, we report the strategy for producing 3HP and its copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) by the development of H. bluephagenesis. The transcriptome analysis reveals its 3HP degradation and synthesis pathways involving endogenous synthetic enzymes from 1,3-propanediol. Combing the optimized expression of aldehyde dehydrogenase (AldDHb), an engineered H. bluephagenesis strain of whose 3HP degradation pathway is deleted and that overexpresses alcohol dehydrogenases (AdhP) on its genome under a balanced redox state, is constructed with an enhanced 1.3-propanediol-dependent 3HP biosynthetic pathway to produce 154 g L-1 of 3HP with a yield and productivity of 0.93 g g-1 1,3-propanediol and 2.4 g L-1 h-1, respectively. Moreover, the strain could also accumulate 60% poly(3-hydroxybutyrate-co-32-45% 3-hydroxypropionate) in the dry cell mass, demonstrating to be a suitable chassis for hyperproduction of 3HP and P3HB3HP.


Asunto(s)
Vías Biosintéticas , Halomonas/genética , Halomonas/metabolismo , Ácido Láctico/análogos & derivados , Ácido Láctico/biosíntesis , Ingeniería Metabólica , Proteínas Bacterianas/metabolismo , Biopolímeros/metabolismo , Vías Biosintéticas/genética , Edición Génica , Regulación Bacteriana de la Expresión Génica , Halomonas/enzimología , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Glicoles de Propileno/metabolismo
20.
Nat Commun ; 12(1): 1625, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712575

RESUMEN

Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. Here we report that in the gastrointestinal pathogens Yersinia enterocolitica and Shigella flexneri, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. We show that in Yersinia enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the T3SS at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates T3SS activity in response to environmental conditions.


Asunto(s)
Citosol/metabolismo , Transporte de Proteínas/fisiología , Sistemas de Secreción Tipo III/metabolismo , Adhesión Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , Shigella flexneri/metabolismo , Sistemas de Secreción Tipo III/genética , Yersinia enterocolitica/metabolismo
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