The Pangenome of Gram-Negative Environmental Bacteria Hides a Promising Biotechnological Potential.

Secondary metabolites (SMs) from environmental bacteria offer viable solutions for various health and environmental challenges. Researchers are employing advanced bioinformatic tools to investigate less-explored microorganisms and unearth novel bioactive compounds. In this research area, our understanding of SMs from environmental Gram-negative bacteria lags behind that of its Gram-positive counterparts. In this regard, Pedobacter spp. have recently gained attention, not only for their role as plant growth promoters but also for their potential in producing antimicrobials. This study focuses on the genomic analysis of Pedobacter spp. to unveil the diversity of the SMs encoded in their genomes. Among the 41 genomes analyzed, a total of 233 biosynthetic gene clusters (BGCs) were identified, revealing the potential for the production of diverse SMs, including RiPPs (27%), terpenes (22%), hybrid SMs (17%), PKs (12%), NRPs (9%) and siderophores (6%). Overall, BGC distribution did not correlate with phylogenetic lineage and most of the BGCs showed no significant hits in the MIBiG database, emphasizing the uniqueness of the compounds that Pedobacter spp. can produce. Of all the species examined, P. cryoconitis and P. lusitanus stood out for having the highest number and diversity of BGCs. Focusing on their applicability and ecological functions, we investigated in greater detail the BGCs responsible for siderophore and terpenoid production in these species and their relatives. Our findings suggest that P. cryoconitis and P. lusitanus have the potential to produce novel mixtures of siderophores, involving bifunctional IucAC/AcD NIS synthetases, as well as carotenoids and squalene. This study highlights the biotechnological potential of Pedobacter spp. in medicine, agriculture and other industries, emphasizing the need for a continued exploration of its SMs and their applications.

The Unexplored Wealth of Microbial Secondary Metabolites: the Sphingobacteriaceae Case Study.

Research on secondary metabolites (SMs) has been mostly focused on Gram-positive bacteria, especially Actinobacteria. The association of genomics with robust bioinformatics tools revealed the neglected potential of Gram-negative bacteria as promising sources of new SMs. The family Sphingobacteriaceae belongs to the phylum Bacteroidetes having representatives in practically all environments including humans, rhizosphere, soils, wastewaters, among others. Some genera of this family have demonstrated great potential as plant growth promoters, bioremediators and producers of some value-added compounds such as carotenoids and antimicrobials. However, to date, Sphingobacteriaceae's SMs are still poorly characterized, and likewise, little is known about their chemistry. This study revealed that Sphingobacteriaceae pangenome encodes a total of 446 biosynthetic gene clusters (BGCs), which are distributed across 85 strains, highlighting the great potential of this bacterial family to produce SMs. Pedobacter, Mucilaginibacter and Sphingobacterium were the genera with the highest number of BGCs, especially those encoding the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), terpenes, polyketides and nonribosomal peptides (NRPs). In Mucilaginibacter and Sphingobacterium genera, M. lappiensis ATCC BAA-1855, Mucilaginibacter sp. OK098 (both with 11 BGCs) and Sphingobacterium sp. 21 (6 BGCs) are the strains with the highest number of BGCs. Most of the BGCs found in these two genera did not have significant hits with the MIBiG database. These results strongly suggest that the bioactivities and environmental functions of these compounds, especially RiPPs, PKs and NRPs, are still unknown. Among RiPPs, two genera encoded the production of class I and class III lanthipeptides. The last are associated with LanKC proteins bearing uncommon lyase domains, whose dehydration mechanism deserves further investigation. This study translated genomics into functional information that unveils the enormous potential of environmental Gram-negative bacteria to produce metabolites with unknown chemistries, bioactivities and, more importantly, unknown ecological roles.

Peptone from casein, an antagonist of nonribosomal peptide synthesis: a case study of pedopeptins produced by Pedobacter lusitanus NL19.

Novel natural products are urgently needed to address the worldwide incidence of bacterial resistance to antibiotics. Extreme environments are a major source of novel compounds with unusual chemical structures. Pedobacter lusitanus NL19 is a new bacterial species that was isolated from one such environment and which produces compounds with potent activity against relevant microorganisms in the clinical, food, veterinary and aquaculture areas. The production of antimicrobials by P. lusitanus NL19 was identified in tryptic soy agar (TSA), but not in its equivalent broth (TSB). It was observed that in TSB medium a high concentration of casein peptone (PC) repressed the production of antibacterial compounds. HPLC, MS and MS/MS spectra with de novo sequencing revealed that the bioactivity of P. lusitanus NL19 was due to the production of pedopeptins. Hence, biosynthesis of pedopeptins is inhibited by high concentrations of PC in the broth medium. Furthermore, a nonribosomal peptide synthetase (NRPS) gene cluster was identified in the genome of NL19 encoding the biosynthesis of the peptides. qPCR analysis confirmed that the transcription of these genes is repressed in cells cultivated in high concentrations of PC. It is shown that pedopeptins are nonribosomal peptides with a broad-spectrum activity, including against Gram-positive and Gram-negative bacteria and yeasts.

Environmental superbugs: The case study of Pedobacter spp.

The environment is one of the main reservoirs of antibiotic resistance genes (ARGs) but multidrug resistant (MDR) environmental isolates are barely characterised. As suggested by the name, Pedobacter species have been predominantly isolated from soils, but are also recovered from water (including drinking water), chilled food, fish, compost, sludge, glaciers and other extreme environments. The susceptibility phenotype of Pedobacter lusitanus NL19 (isolated from a deactivated uranium mine), its closely related species and the genus type strain were investigated. All strains are MDR bacteria, resistant to ß-lactams, colistin, aminoglycosides and ciprofloxacin. Therefore, Pedobacter spp. are likely intrinsically resistant to ß-lactams (including ertapenem) and to other three classes of antibiotics. 6%-8% of their total protein-encoding genes encode a diverse collection of putative ARGs, including ß-lactamases. These enzymes are highly abundant in all the other Pedobacter strains with sequenced genomes, especially class C, class B3 and class A. LUS-1 and PLN-1 were further characterised in E. coli. LUS-1 is a class A ß-lactamase and it conferred an increase in the MIC of cefotaxime, albeit very low. PLN-1 is a class B3 ß-lactamase with carbapenemase activity, conferring resistance to ertapenem and a 66x and 16x increase in the MIC of imipenem and meropenem, respectively. PLN-1 also hydrolyses ampicillin, 1st and 3rd generation cephalosporins, and at a lower extent cephamycins and 4th generation cephalosporins. Therefore, Pedobacter spp. encode a large and diverse arsenal of resistance mechanisms that make them environmental superbugs.

Pedobacter lusitanus sp. nov., isolated from sludge of a deactivated uranium mine.

Strain NL19T is a Gram-stain-negative, aerobic bacterium that was isolated from sludge of a deactivated uranium mine in Portugal. 16S rRNA gene sequence analysis revealed that strain NL19T is a member of the genus Pedobacter and closely related to the strains Pedobacter himalayensis MTCC 6384T, Pedobacter cryoconitis DSM 14825T, Pedobacter westerhofensis DSM 19036T and Pedobacterhartonius DSM 19033T. It had a DNA G+C content of 40.8 mol%, which agreed with the genus description. The main fatty acids included C16 : 1ω7c, C14 : 1ω5c, C4 : 0, iso-C17 : 0, iso-C17 : 0 3-OH, C16 : 0, anteiso-C15 : 0 and iso-C15 : 0 3-OH. The main lipids present were phospholipids (60 %) and sphingolipids (35 %). The most abundant phospholipids included phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine. Menaquinone-7 (MK-7) was the only isoprenoid quinone detected. DNA-DNA hybridization similarities between strain NL19T and Pedobacter himalayensis MTCC 6384T, Pedobacter cryoconitis DSM 14825T, Pedobacter westerhofensis DSM 19036T and Pedobacter hartonius DSM 19033T were 15.3 , 16.2 , 11.5 and 16.0 %, respectively. Strain NL19T can also be distinguished from these four species based on gyrB and intergenic transcribed spacers (ITS) sequences and by some phenotypic traits such as NaCl tolerance, pH, growth temperature and carbon source utilization. Strain NL19Trepresents a novel species of the genus Pedobacter, for which the name Pedobacter lusitanus sp. nov. is proposed. The type strain is NL19T (=LMG 29220T=CECT 9028T). An amended description of Pedobacter himalayensis is also included.

Draft Genome Sequence of Pedobacter sp. Strain NL19, a Producer of Potent Antibacterial Compounds.

Here, we report the draft genome sequence of Pedobacter sp. strain NL19. The genome has 5.99 Mbp and a G+C content of 39.0%. NL19 was isolated from sludge from an abandoned uranium mine in the north of Portugal, and it produces potent antibacterials against Gram-positive and Gram-negative bacteria.

Pre-miR-146a (rs2910164 G>C) single nucleotide polymorphism is genetically and functionally associated with leprosy.

Mycobacterium leprae infects macrophages and Schwann cells inducing a gene expression program to facilitate its replication and progression to disease. MicroRNAs (miRNAs) are key regulators of gene expression and could be involved during the infection. To address the genetic influence of miRNAs in leprosy, we enrolled 1,098 individuals and conducted a case-control analysis in order to study four miRNAs genes containing single nucleotide polymorphism (miRSNP). We tested miRSNP-125a (rs12975333 G>T), miRSNP-223 (rs34952329 *>T), miRSNP-196a-2 (rs11614913 C>T) and miRSNP-146a (rs2910164 G>C). Amongst them, miRSNP-146a was the unique gene associated with risk to leprosy per se (GC OR = 1.44, p = 0.04; CC OR = 2.18, p = 0.0091). We replicated this finding showing that the C-allele was over-transmitted (p = 0.003) using a transmission-disequilibrium test. A functional analysis revealed that live M. leprae (MOI 100:1) was able to induce miR-146a expression in THP-1 (p<0.05). Furthermore, pure neural leprosy biopsies expressed augmented levels of that miRNA as compared to biopsy samples from neuropathies not related with leprosy (p = 0.001). Interestingly, carriers of the risk variant (C-allele) produce higher levels of mature miR-146a in nerves (p = 0.04). From skin biopsies, although we observed augmented levels of miR-146a, we were not able to correlate it with a particular clinical form or neither host genotype. MiR-146a is known to modulate TNF levels, thus we assessed TNF expression (nerve biopsies) and released by peripheral blood mononuclear cells infected with BCG Moreau. In both cases lower TNF levels correlates with subjects carrying the risk C-allele, (p = 0.0453 and p = 0.0352; respectively), which is consistent with an immunomodulatory role of this miRNA in leprosy.

Pre-miR 146a (rs2910164 G>C) single nucleotide polymorphism is genetically and functionally associated with leprosy

Mycobacterium leprae infects macrophages and Schwann cells inducing a gene expression program to facilitate its replication and progression to disease. MicroRNAs (miRNAs) are key regulators of gene expression and could be involved during the infection. To address the genetic influence of miRNAs in leprosy, we enrolled 1,098 individuals and conducted a case-control analysis in order to study four miRNAs genes containing single nucleotide polymorphism (miRSNP). We tested miRSNP-125a (rs12975333 G>T), miRSNP-223 (rs34952329 *>T), miRSNP-196a-2 (rs11614913 C>T) and miRSNP-146a (rs2910164 G>C). Amongst them, miRSNP-146a was the unique gene associated with risk to leprosy per se (GC OR = 1.44, p = 0.04; CC OR = 2.18, p = 0.0091). We replicated this finding showing that the C-allele was over-transmitted (p = 0.003) using a transmission-disequilibrium test. A functional analysis revealed that live M. leprae (MOI 100:1) was able to induce miR-146a expression in THP-1 (p<0.05). Furthermore, pure neural leprosy biopsies expressed augmented levels of that miRNA as compared to biopsy samples from neuropathies not related with leprosy (p = 0.001). Interestingly, carriers of the risk variant (C-allele) produce higher levels of mature miR-146a in nerves (p = 0.04). From skin biopsies, although we observed augmented levels of miR-146a, we were not able to correlate it with a particular clinical form or neither host genotype. MiR-146a is known to modulate TNF levels, thus we assessed TNF expression (nerve biopsies) and released by peripheral blood mononuclear cells infected with BCG Moreau. In both cases lower TNF levels correlates with subjects carrying the risk C-allele, (p = 0.0453 and p = 0.0352; respectively), which is consistent with an immunomodulatory role of this miRNA in leprosy.

Leishmania braziliensis-reactive T cells are down-regulated in long-term cured cutaneous Leishmaniasis, but the renewal capacity of T effector memory compartments is preserved.

Leishmania (Viannia) braziliensis control and tissue damage relate to the effector immune response, which in turn affects clinical outcome. Leishmania reactive CD4(+) and CD8(+) T cells are expanded in long-term healed cutaneous leishmaniasis (hCL) patients but their functional characteristics remain to be determined. This study investigates antigen-specific recall in long-term healed CL caused by L. braziliensis infection. Healed CL subjects were grouped according to the time elapsed since the end of therapy: less than two years and two to five years. Activation phenotype (CD69(+) or CD25(+)) and subpopulations of memory T cell phenotypes [central memory (Tcm): CD45RO(+) CCR7(+) or effector memory (Tem): CD45RO(+) CCR7(-)] were quantified in ex vivo blood mononuclear cells and after Leishmania antigens stimuli. A reduction in the percentage of activated Leishmania-responder CD4(+) and CD8(+) T cells in hCL was associated with the time elapsed since clinical cure. Percentage of CD69(+) in TCD4(+) and TCD8(+) cells were negatively correlated with IL-10 levels. Ex vivo analyses showed contracted Tem CD4(+) and Tem CD8(+) compartments from hCL with long time elapsed since clinical cure, although renewal of these compartments was observed following in vitro exposure to leishmanial stimuli. Our results show that healed L. braziliensis infected patients exhibit a recall response to Leishmania antigens with evident expansion of effector memory T cells. Regulated leishmanial-specific response seems to emerge only about two years after initial contact with the parasite antigens.

Candidate gene case-control and functional study shows macrophage inhibitory factor (MIF) polymorphism is associated with cutaneous leishmaniasis.

American tegumentary leishmaniasis (ATL) is an infectious disease caused mostly by Leishmania(Viannia)braziliensis in Southeast Brazil. The clinical manifestations are vast, ranging from asymptomatic to severe mucosal leishmaniasis (ML). It has been suggested that variation of the pathogen does not fully explain the response spectrum and the variability of clinical manifestations. Previous data have shown that host genetics also play a role in disease outcome. Herein, we have tested the association of TNF, IL10, IL12 and MIF single nucleotide polymorphisms (SNPs) using a case-control study design including 110 cutaneous leishmaniasis (CL) patients and 682 healthy subjects. The genotype-phenotype correlation was also assessed using leishmania antigens to stimulate peripheral blood mononuclear cells obtained from cured CL patients. Results demonstrated that the MIF -173C allele is associated with leishmaniasis outcome and also with lower levels of MIF in culture supernatants. Also, the TNF -308AA genotype was statistically increased among leishmaniasis patients. The results showed here suggest that the lower levels of MIF produced by MIF -173C carriers could influence the host-Leishmania interaction, favoring infection and disease progression. On the other hand, high TNF levels can contribute to tissue damage, consequently leading to skin lesions.

Estudo da influência de polimorfismos nos genes IL-10, IL-12, MIF e TNF na imunopatogênese da leishmaniose tegumentar americana / The influence of polymorphisms in IL-10, IL-12, MIF and TNF in the immunopathogenesis of leishmaniasis

A leishmaniose tegumentar Americana (LTA) representa um conjunto de doenças com características clínicas e imunopatológicas distintas, cuja evolução depende, pelo menos em parte, de fatores relacionados à resposta imune do hospedeiro. A imunopatogênese da leishmaniose mucosa (LM), causadas principalmente pela L. braziliensis, é dependente de uma intensa resposta celular do tipo 1, dirigida principalmente pela elevada secreção das citocinas IFN-gama e TNF, que apresentam expressão aumentada em presença de IL-12. Por outro lado, várias evidências apontam que a IL-10 é a principal citocina envolvida na regulação da resposta imune em pacientes com leishmaniose, geralmente suprimindo a imunidade mediada por células. Estudos recentes vêm demonstrando que a susceptibilidade ou resistência a dor do hospedeiro a patógenos intracelulares, bem como a gravidade a várias doenças infecciosas podem ser associados com polimorfismos em genes de citocinas. Assim, com o propósito de estabelecer marcadores que possuam ter influência genética na evolução clínica da LTA, o presente trabalho teve como objetivo avaliar as frequências dos SNPs nos genes da IL-10 - 819C maior que T, IL-12 -1188 maior que C 3 UTR, MIF -173G maior que C e TNF - 308G maior que A em pacientes com histórico de LTA e controles saudáveis. Além disso, buscou-se analisar a associação dos genótipos com a produção das citocinas IL-10, IL-12p70, MIF e TNF nas diferentes formas clínicas da LTA e nos controles saudáveis. No que se refere aos SNPs IL-10 -819 e IL-12 +1188 não foi observada uma associação dos mesmos com a LTA per se, no entanto, os SNP MIF -17 e TNF -308 sugerem risco à doença: MIF -173GC (OR=0,04; p=0,04) e TNF – 308AA (OR=3,92; p=0,0001). Nenhum dos SNPs estudados apresentou relação com a progressão para a LM, considerada a forma mais grave da LTA. Em relação à dosagem das citocinas IL-10, IL-12, MIF e TNF os indivíduos controles apresentaram níveis menores do que os pacientes...

IFNG +874T/A polymorphism is not associated with American tegumentary leishmaniasis susceptibility but can influence Leishmania induced IFN-gamma production.

BACKGROUND: Interferon-gamma is a key cytokine in the protective responses against intracellular pathogens. A single nucleotide polymorphism (SNP) located in the first intron of the human IFN-gamma gene can putatively influence the secretion of cytokine with an impact on infection outcome as demonstrated for tuberculosis and other complex diseases. Our aim was to investigate the putative association of IFNG+874T/A SNP with American tegumentary leishmaniasis (ATL) and also the influence of this SNP in the secretion of IFN-gamma in vitro. METHODS: Brazilian ATL patients (78 cutaneous, CL, and 58 mucosal leishmaniasis, ML) and 609 healthy volunteers were evaluated. The genotype of +874 region in the IFN-gamma gene was carried out by Amplification Refractory Mutational System (ARMS-PCR). Leishmania-induced IFN-gamma production on peripheral blood mononuclear cell (PBMC) culture supernatants was assessed by ELISA. RESULTS: There are no differences between +874T/A SNP frequency in cases and controls or in ML versus CL patients. Cutaneous leishmaniasis cases exhibiting AA genotype produced lower levels of IFN-gamma than TA/TT genotypes. In mucosal cases, high and low IFN-gamma producers were clearly demonstrated but no differences in the cytokine production was observed among the IFNG +874T or A carriers. CONCLUSION: Our results suggest that +874T/A polymorphism was not associated with either susceptibility or severity to leishmaniasis. Despite this, IFNG +874T/A SNP could be involved in the pathogenesis of leishmaniasis by influencing the amount of cytokine released by CL patients, although it could not prevent disease development. On the other hand, it is possible that in ML cases, other potential polymorphic regulatory genes such as TNF-alpha and IL-10 are also involved thus interfering with IFN-gamma secretion.