Genetically engineered Lactococcus lactis strain constitutively expresses GABA-producing genes and produces high levels of GABA.

γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter of the central nervous system that impacts physical and mental health. Low GABA levels have been documented in several diseases, including multiple sclerosis and depression, and studies suggest that GABA could improve disease outcomes in those conditions. Probiotic bacteria naturally produce GABA and have been engineered to enhance its synthesis. Strains engineered thus far use inducible expression systems that require the addition of exogenous molecules, which complicates their development as therapeutics. This study aimed to overcome this challenge by engineering Lactococcus lactis with a constitutive GABA synthesis gene cassette. GABA synthesizing and transport genes (gadB and gadC) were cloned onto plasmids downstream of constitutive L. lactis promoters [P2, P5, shortened P8 (P8s)] of different strengths and transformed into L. lactis. Fold increase in gadCB expression conferred by these promoters (P2, P5, and P8s) was 322, 422, and 627, respectively, compared to the unmodified strain (P = 0.0325, P8s). GABA synthesis in the highest gadCB expressing strain, L. lactis-P8s-glutamic acid decarboxylase (GAD), was dependent on media supplementation with glutamic acid and significantly higher than the unmodified strain (P < 0.0001, 125 mM, 200 mM glutamic acid). Lactococcus lactis-P8s-GAD is poised for therapeutic testing in animal models of low-GABA-associated disease.

The Microbiome as a Therapeutic Target for Multiple Sclerosis: Can Genetically Engineered Probiotics Treat the Disease?

There is an increasing interest in the intestinal microbiota as a critical regulator of the development and function of the immune, nervous, and endocrine systems. Experimental work in animal models has provided the foundation for clinical studies to investigate associations between microbiota composition and function and human disease, including multiple sclerosis (MS). Initial work done using an animal model of brain inflammation, experimental autoimmune encephalomyelitis (EAE), suggests the existence of a microbiota-gut-brain axis connection in the context of MS, and microbiome sequence analyses reveal increases and decreases of microbial taxa in MS intestines. In this review, we discuss the impact of the intestinal microbiota on the immune system and the role of the microbiome-gut-brain axis in the neuroinflammatory disease MS. We also discuss experimental evidence supporting the hypothesis that modulating the intestinal microbiota through genetically modified probiotics may provide immunomodulatory and protective effects as a novel therapeutic approach to treat this devastating disease.

Resistencia y sensibilidad bacteriana en urocultivos en una población de mujeres de ecuador / Bacterial resistance and sensitivity in urine cultures in a women population in ecuador / Resistência e sensibilidade bacteriana em uroculturas numa população de mulheres do equador

Resumen Objetivo: Determinar la prevalencia de uropatógenos, sensibilidad y resistencia antimicrobiana en la infección del tracto urinario que acuden al Hospital Básico Privado "Provida" del 1 de enero de 2014 al 31 de diciembre de 2016. Material y métodos: Se analizaron los resultados de 116 urocultivos de orina en mujeres no gestantes de todas las edades de 2014 a 2016, que fueron atendidas en el Hospital Básico Privado "Provida" de la cuidad de Latacunga, en Ecuador. El análisis de los datos obtenidos se realizó mediante estadística descriptiva. Resultados: De las 116 muestras, se aislaron: Escherichia coli (84,5%), Staphylococcus saprophyticus (8,6%) y Proteus spp. (6,9%). E. coli mostró sensibilidad a ceftriaxona en el 70 %, seguido de fosfomicina y gentamicina con el 62 y el 60%, respectivamente. La sensibilidad hallada para quinolonas fue del 40% y la ampicilina sulbactam alcanzó el 37%. Proteus spp. mostró sensibilidad del 75% para gentamicina y del 50% para quinolonas y cefuroxima. S. saprophyticus tuvo sensibilidad superior al 50% para gentamicina, ampicilina sulbactam, quinolonas y nitrofurantoína. Para E. coli la resistencia más alta registrada fue con ampicilina en el 86,5%, seguido de las quinolonas con una resistencia superior al 50%. La ampicilina asociada a inhibidor de betalactamasas, fosfomicina, cefalosporinas, nitrofurantoína y aminoglucósidos mostró resistencia inferior al 25%. Conclusión: El agente patógeno más prevalente en infecciones del tracto urinario (ITU) es E. coli (84,7%), porcentaje coincidente con lo reportado en la literatura nacional y mundial. Los antimicrobianos para este uropatógeno con mayor resistencia fueron ampicilina (86%), cirprofloxacina (55%) y norfloxacina (53%). Se podría tener en cuenta en el momento de administrar una terapéutica empírica, dato que debería ser corroborado con información de susceptibilidades de acuerdo con el contexto.

Summary Objective: To determine the prevalence of uropathogens, sensitivity and antimicrobial resistance in urinary tract infections that go to the Private Basic Hospital "Provida" from January 1, 2014 to December 31, 2016. Material and methods: The results of 116 urine cultures in non-pregnant women of all ages from 2014 to 2016, which were treated at the Private Basic Hospital "Provida" of the city of Latacunga, in Ecuador, were analyzed. The data obtained was analyzed using descriptive statistics. Results: In the 116 samples, Escherichia coli (84.5%), Staphylococcus saprophyticus (8.6%) and Proteus spp. (6.9%) were isolated. E. coli showed sensitivity to ceftriaxone in 70%, followed by fosfomycin and gentamicin with 62 and 60%, respectively. The sensitivity found for quinolones was 40% and for sulbactam ampicillin reached a 37%. Proteus spp. showed sensitivity of 75% for gentamicin and 50% for quinolones and cefuroxime. S. saprophyticus had a sensitivity greater than 50% for gentamicin, sulbactam ampicillin, quinolones and nitrofurantoin. For E. coli the highest resistance recorded was found on ampicillin in 86.5%, followed by quinolones with a resistance greater than 50%. Ampicillin associated with inhibitor of beta-lactamase, fosfomycin, cephalosporins, nitrofurantoin and aminoglycosides showed a resistance below 25%. Conclusion: The most prevalent pathogen in urinary tract infections (UTI) is E. coli (84.7%), a percentage that matches what has been reported in national and world literature. The antimicrobials for this uropathogen with the highest resistance were ampicillin (86%), ciprofloxacin (55%) and norfloxacin (53%). This should be taken into account when administering an empiric therapy, even though this data should be corroborated with the susceptibility information depending on the context.

Resumo Objetivo: Determinar a prevalência de uropatógenos, sensibilidade e resistência antimicrobiana na infecção do trato urinário que vão ao Hospital Básico Privado "Provida" de 1 de janeiro de 2014 a 31 de dezembro de 2016. Material e métodos: Analisaram-se os resultados de 116 uroculturas de urina em mulheres não gestantes de todas as idades de 2014 a 2016, que foram atendidas no Hospital Básico Privado "Provida" da cidade de Latacunga, no Equador. A análise dos dados obtidos realizou-se mediante estatística descritiva. Resultados: Das 116 amostras, isolaram-se: Escherichia coli (84,5%), Staphylococcus saprophyticus (8,6%) e Proteus spp. (6,9%). E. coli mostrou sensibilidade a ceftriaxona em 70 %, seguido de fosfomicina e gentamicina com 62 e 60%, respectivamente. A sensibilidade encontrada para quinolonas foi de 40% e a ampicilina sulbactam atingiu 37%. Proteus spp. mostrou sensibilidade de 75% para gentamicina e de 50% para quinolonas e cefuroxima. S. saprophyticus teve sensibilidade superior a 50% para gentamicina, ampicilina sulbac-tam, quinolonas e nitrofurantoína. Para E. coli a resistência mais alta registrada foi com ampicilina em 86,5%, seguido das quinolonas com uma resistência superior a 50%. A ampicilina associada a inibidor de betalactamasas, fosfomicina, cefalosporinas, nitrofurantoína e aminoglucósidos mostrou resistência inferior a 25%. Conclusão: O agente patogénico mais prevalente em infecções do trato urinário (ITU) é E. coli (84,7%), porcentagem coincidente com o apresentado na literatura nacional e mundial. Os antimicrobianos para esse uropatógeno com maior resistência foram ampicilina (86%), cirprofloxacina (55%) e norfloxacina (53%). Poderia ser tido em conta no momento de administrar uma terapêutica empírica, dado que deveria ser corroborado com informação de suscetibilidades de acordo com o contexto.

Conserved transcriptional unit organization of the cag pathogenicity island among Helicobacter pylori strains.

The Helicobacter pyloricag pathogenicity island (cag PAI) encodes a type IV secretion system that is more commonly found in strains isolated from patients with gastroduodenal disease than from those with asymptomatic gastritis. Genome-wide organization of the transcriptional units in H. pylori strain 26695 was recently established using RNA sequence analysis (Sharma et al., 2010). Here we used quantitative reverse-transcription polymerase chain reaction of open reading frames and intergenic regions to identify putative cag PAI operons in H. pylori; these operons were analyzed further by transcript profiling after deletion of selected promoter regions. Additionally, we used a promoter-trap system to identify functional cag PAI promoters. The results demonstrated that expression of genes on the H. pyloricag PAI varies by nearly five orders of magnitude and that the organization of cag PAI genes into transcriptional units is conserved among several H. pylori strains, including, 26695, J99, G27, and J166. We found evidence for 20 transcripts within the cag PAI, many of which likely overlap. Our data suggests that there are at least 11 operons: cag1-4, cag3-4, cag10-9, cag8-7, cag6-5, cag11-12, cag16-17, cag19-18, cag21-20, cag23-22, and cag25-24, as well as five monocistronic genes (cag4, cag13, cag14, cag15, and cag26). Additionally, the location of four of our functionally identified promoters suggests they are directing expression of, in one case, a truncated version of cag26 and in the other three, transcripts that are antisense to cag7, cag17, and cag23. We verified expression of two of these antisense transcripts, those antisense to cag17 and cag23, by reverse-transcription polymerase chain reaction. Taken together, our results suggest that the cag PAI transcriptional profile is generally conserved among H. pylori strains, 26695, J99, G27, and J166, and is likely complex.

The Helicobacter pylori autotransporter ImaA (HP0289) modulates the immune response and contributes to host colonization.

The human pathogen Helicobacter pylori employs a diverse collection of outer membrane proteins to colonize, persist, and drive disease within the acidic gastric environment. In this study, we sought to elucidate the function of the host-induced gene HP0289, which encodes an uncharacterized outer membrane protein. We first generated an isogenic H. pylori mutant that lacks HP0289 and found that the mutant has a colonization defect in single-strain infections and is greatly outcompeted in mouse coinfection experiments with wild-type H. pylori. Furthermore, we used protease assays and biochemical fractionation coupled with an HP0289-targeted peptide antibody to verify that the HP0289 protein resides in the outer membrane. Our previous findings showed that the HP0289 promoter is upregulated in the mouse stomach, and here we demonstrate that HP0289 expression is induced under acidic conditions in an ArsRS-dependent manner. Finally, we have shown that the HP0289 mutant induces greater expression of the chemokine interleukin-8 (IL-8) and the cytokine tumor necrosis factor alpha (TNF-α) in gastric carcinoma cells (AGS). Similarly, transcription of the IL-8 homolog keratinocyte-derived chemokine (KC) is elevated in murine infections with the HP0289 mutant than in murine infections with wild-type H. pylori. On the basis of this phenotype, we renamed HP0289 ImaA for immunomodulatory autotransporter protein. Our work has revealed that genes induced in vivo play an important role in H. pylori pathogenesis. Specifically, the outer membrane protein ImaA modulates a component of the host inflammatory response, and thus may allow H. pylori to fine tune the host immune response based on ImaA expression.

Recombination-based in vivo expression technology identifies Helicobacter pylori genes important for host colonization.

Here we undertook to identify colonization and gastric disease-promoting factors of the human gastric pathogen Helicobacter pylori as genes that were induced in response to the stomach environment. Using recombination-based in vivo expression technology (RIVET), we identified six promoters induced in the host compared to laboratory conditions. Three of these promoters, designated Pivi10, Pivi66, and Pivi77, regulate genes that H. pylori may use to interact with other microbes or the host. Pivi10 likely regulates the mobA, mobB, and mobD genes, which have potential roles in horizontal gene transfer through plasmid mobilization. Pivi66 occurs in the cytotoxin-associated gene pathogenicity island, a genomic region known to be associated with more severe disease outcomes, and likely regulates cagZ, virB11, and virD4. Pivi77 likely regulates HP0289, an uncharacterized paralogue of the vacA cytotoxin gene. We assessed the roles of a subset of these genes in colonization by creating deletion mutants and analyzing them in single-strain and coinfection experiments. We found that a mobABD mutant was defective for murine host colonization and that a cagZ mutant outcompeted the wild-type strain in a coinfection analysis. Our work supports the conclusion that RIVET is a valuable tool for identifying H. pylori factors with roles in host colonization.

Experimental analysis of Helicobacter pylori transcriptional terminators suggests this microbe uses both intrinsic and factor-dependent termination.

In this study, we report experimental analysis of transcriptional terminators in the human pathogen Helicobacter pylori. Previous bioinformatics approaches came to differing conclusions regarding transcriptional termination in this bacterium. We used a reporter construct, the tnpR-encoded resolvase, to assess terminators. In our first experiments, we found that a subset of previously predicted intrinsic terminators for H. pylori are functional. In our second experiments, we used an unbiased screen to identify putative terminators and then characterized 18 of these. We found that these putative terminators overlap genomic regions that are either intergenic or intragenic. Using reverse transcription PCR, we showed that an intergenic terminator and an intragenic antisense terminator function at their endogenous loci. Additionally, we found that putative terminators contain features of both intrinsic and Rho-dependent termination, but that intrinsic terminators define the majority. We were unable to delete rho, however, in H. pylori, suggesting that it is essential and likely important. Finally, we carried out a mutational analysis of one of our randomly identified terminators that has both intrinsic and Rho-dependent features, and found that they are both functional. In conclusion, we found that H. pylori possesses numerous Rho-dependent and intrinsic terminators including some found in intragenic regions.

De novo kinetochore assembly requires the centromeric histone H3 variant.

Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.

The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p.

Saccharomyces cerevisiae MPS1 encodes an essential protein kinase that has roles in spindle pole body (SPB) duplication and the spindle checkpoint. Previously characterized MPS1 mutants fail in both functions, leading to aberrant DNA segregation with lethal consequences. Here, we report the identification of a unique conditional allele, mps1-8, that is defective in SPB duplication but not the spindle checkpoint. The mutations in mps1-8 are in the noncatalytic region of MPS1, and analysis of the mutant protein indicates that Mps1-8p has wild-type kinase activity in vitro. A screen for dosage suppressors of the mps1-8 conditional growth phenotype identified the gene encoding the integral SPB component SPC42. Additional analysis revealed that mps1-8 exhibits synthetic growth defects when combined with certain mutant alleles of SPC42. An epitope-tagged version of Mps1p (Mps1p-myc) localizes to SPBs and kinetochores by immunofluorescence microscopy and immuno-EM analysis. This is consistent with the physical interaction we detect between Mps1p and Spc42p by coimmunoprecipitation. Spc42p is a substrate for Mps1p phosphorylation in vitro, and Spc42p phosphorylation is dependent on Mps1p in vivo. Finally, Spc42p assembly is abnormal in a mps1-1 mutant strain. We conclude that Mps1p regulates assembly of the integral SPB component Spc42p during SPB duplication.