Elimination of PknL and MSMEG_4242 in Mycobacterium smegmatis alters the character of the outer cell envelope and selects for mutations in Lsr2.

Four serine/threonine kinases are present in all mycobacteria: PknA, PknB, PknG and PknL. PknA and PknB are essential for growth and replication, PknG regulates metabolism, but little is known about PknL. Inactivation of pknL and adjacent regulator MSMEG_4242 in rough colony M. smegmatis mc2155 produced both smooth and rough colonies. Upon restreaking rough colonies, smooth colonies appeared at a frequency of ~ 1/250. Smooth mutants did not form biofilms, showed increased sliding motility and anomalous lipids on thin-layer chromatography, identified by mass spectrometry as lipooligosaccharides and perhaps also glycopeptidolipids. RNA-seq and Sanger sequencing revealed that all smooth mutants had inactivated lsr2 genes due to mutations and different IS1096 insertions. When complemented with lsr2, the colonies became rough, anomalous lipids disappeared and sliding motility decreased. Smooth mutants showed increased expression of IS1096 transposase TnpA and MSMEG_4727, which encodes a protein similar to PKS5. When MSMEG_4727 was deleted, smooth pknL/MSMEG_4242/lsr2 mutants reverted to rough, formed good biofilms, their motility decreased slightly and their anomalous lipids disappeared. Rough delpknL/del4242 mutants formed poor biofilms and showed decreased, aberrant sliding motility and both phenotypes were complemented with the two deleted genes. Inactivation of lsr2 changes colony morphology from rough to smooth, augments sliding motility and increases expression of MSMEG_4727 and other enzymes synthesizing lipooligosaccharides, apparently preventing biofilm formation. Similar morphological phase changes occur in other mycobacteria, likely reflecting environmental adaptations. PknL and MSMEG_4242 regulate lipid components of the outer cell envelope and their absence selects for lsr2 inactivation. A regulatory, phosphorylation cascade model is proposed.

ß-defensin-2 in breast milk displays a broad antimicrobial activity against pathogenic bacteria / ß-defensina 2 no leite materno mostra uma ampla atividade antimicrobiana contra bactérias patogênicas

OBJECTIVE: To describe the antimicrobial activity of ß-defensin-2 produced in the mammary gland and secreted in human breast milk. METHODS: The peptide production was performed by DNA cloning. ß-defensin-2 levels were quantified in 61 colostrum samples and 39 mature milk samples from healthy donors, by an indirect enzyme-linked immunosorbent assay (ELISA). Using halo inhibition assay, this study assessed activity against seven clinical isolates from diarrheal feces of children between 0 and 2 years of age. The activity of ß-defensin-2 against three opportunistic pathogens that can cause nosocomial infections was determined by microdilution test. RESULTS: The peptide levels were higher in colostrum (n = 61) than in mature milk samples (n = 39), as follows: median and range, 8.52 (2.6-16.3) µg/ml versus 0.97 (0.22-3.78), p < 0.0001; Mann-Whitney test. The recombinant peptide obtained showed high antimicrobial activity against a broad range of pathogenic bacteria. Its antibacterial activity was demonstrated in a disk containing between 1-4 µg, which produced inhibition zones ranging from 18 to 30 mm against three isolates of Salmonella spp. and four of E. coli. ß-defensin-2 showed minimum inhibitory concentrations (MICs) of 0.25 µg/mL and 0.5 µg/mL for S. marcescen and P. aeruginosa, respectively, while a higher MIC (4 µg/mL) was obtained against an isolated of multidrug-resistant strain of A. baumannii. CONCLUSIONS: To the authors' knowledge, this study is the first to report ß-defensin-2 levels in Latin American women. The production and the activity of ß-defensin-2 in breast milk prove its importance as a defense molecule for intestinal health in pediatric patients. .

OBJETIVO: Descrever a atividade antimicrobiana da defensina-beta 2 na glândula mamária e secretada no leite materno humano. MÉTODOS: A produção de peptídeos foi realizada por clonagem de DNA. Os níveis de defensina-beta 2 foram quantificados em 61 amostras de colostro e 39 de leite maduro de doadoras saudáveis pelo teste ELISA indireto. Por um ensaio de halo de inibição, avaliamos a atividade contra sete isolados clínicos diarreicos de crianças entre 0 e 2 anos. A atividade da defensina 2 contra três patógenos oportunistas que podem causar infecções nosocomiais foi determinada pelo teste de microdiluição. RESULTADOS: Os níveis de peptídeos estavam significativamente maiores nas amostras de colostro (n = 61) que de leite maduro (n = 39), como segue: 8,52 (2,6-16,3 µg/mL) mediana e faixa em comparação a 0,97 (0,22-3,78), p < 0,0001; teste de Mann-Whitney. O peptídeo recombinante foi obtido da alta atividade antimicrobiana demonstrada contra uma ampla gama de bactérias patogênicas. Sua atividade antibacteriana foi demonstrada em um disco contendo entre 1-4 µg, que produziu zonas de inibição entre 18 e 30 mm contra três isolados de Salmonella spp. e quatro de E. coli. A defensina-beta 2 demonstrou concentrações inibitórias mínimas (CIMs) de 0,25 µg/mL e 0,5 µg/mL para S. marcescen and P. aeruginosa, ao passo que uma CIM maior (4 µg/mL) foi obtida contra um isolado de cepa multirresistente de A. baumannii. CONCLUSÕES: Até onde sabemos, este estudo é o primeiro a relatar níveis de defensina em mulheres da América Latina. A produção e a atividade da defensina 2 no leite materno comprovam sua importância como uma molécula de defesa para a saúde intestinal em pacientes pediátricos. .

ß-defensin-2 in breast milk displays a broad antimicrobial activity against pathogenic bacteria.

OBJECTIVE: To describe the antimicrobial activity of ß-defensin-2 produced in the mammary gland and secreted in human breast milk. METHODS: The peptide production was performed by DNA cloning. ß-defensin-2 levels were quantified in 61 colostrum samples and 39 mature milk samples from healthy donors, by an indirect enzyme-linked immunosorbent assay (ELISA). Using halo inhibition assay, this study assessed activity against seven clinical isolates from diarrheal feces of children between 0 and 2 years of age. The activity of ß-defensin-2 against three opportunistic pathogens that can cause nosocomial infections was determined by microdilution test. RESULTS: The peptide levels were higher in colostrum (n=61) than in mature milk samples (n=39), as follows: median and range, 8.52 (2.6-16.3) µg/ml versus 0.97 (0.22-3.78), p<0.0001; Mann-Whitney test. The recombinant peptide obtained showed high antimicrobial activity against a broad range of pathogenic bacteria. Its antibacterial activity was demonstrated in a disk containing between 1-4 µg, which produced inhibition zones ranging from 18 to 30 mm against three isolates of Salmonella spp. and four of E. coli. ß-defensin-2 showed minimum inhibitory concentrations (MICs) of 0.25 µg/mL and 0.5 µg/mL for S. marcescen and P. aeruginosa, respectively, while a higher MIC (4 µg/mL) was obtained against an isolated of multidrug-resistant strain of A. baumannii. CONCLUSIONS: To the authors' knowledge, this study is the first to report ß-defensin-2 levels in Latin American women. The production and the activity of ß-defensin-2 in breast milk prove its importance as a defense molecule for intestinal health in pediatric patients.

Non-pylori Helicobacteraceae in the upper digestive tract of asymptomatic Venezuelan subjects: detection of Helicobacter cetorum-like and Candidatus Wolinella africanus-like DNA.

BACKGROUND: The spectrum of human non-pylori Helicobacter infections is expanding, with species such as H. heilmannii and H. felis occasionally being associated with gastritis. However, the existence of non-pylori Helicobacter colonization in asymptomatic subjects has not been evaluated. The aim of this study was to investigate whether Helicobacter species other than pylori are present in the upper digestive tract of asymptomatic human subjects. MATERIALS AND METHODS: A Helicobacteraceae-specific semi-nested polymerase chain reaction (PCR) assay was used to detect Helicobacter-like organisms in the upper digestive tract of 91 Venezuelan volunteers (aged 18-68 years, 41 females, 50 males). Species were identified by denaturing gradient gel electrophoresis analysis and sequencing of the PCR products. RESULTS: We detected DNA sharing 99-100% sequence identity in over 300-400 bp with the 16S rRNA genes of H. pylori, H. cetorum, and Candidatus Wolinella africanus in 76%, 16%, and 15% of the subjects, respectively. Multiple colonization was documented in 10% of the subjects: H. cetorum and Candidatus W. africanus (4%), H. pylori and Candidatus W. africanus (4%), and H. pylori and H. cetorum (2%). CONCLUSIONS: Our results suggest that non-pylori Helicobacteraceae colonization is relatively common in the Venezuelan asymptomatic population. This is the first report documenting the presence of H. cetorum DNA in the human upper digestive tract, and the second report of the recently discovered Candidatus W. africanus.