Search

Copyright: © 2020 Storey et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.

The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life., This research was supported by the Institute for Tropical Health funders (Obra Social la CAIXA, Fundaciones Caja Navarra and Roviralta, PROFAND, Ubesol, ACUNSA, and Artai) and grants MINECO (AGL2014-58795-C4-1-R, Bru-Epidia 291815- FP7/ERANET/ANIHWA), Aragón Government (Consolidated Group A14), andMarie Curie Career Integration Grant U-KARE (PCIG13-GA-2013-618162). TLB is the recipient of a Ph.D. Fellowship funded by the Department for Employment and Learning (Northern Ireland, United Kingdom).

Using a murine model of Klebsiella pneumoniae bacterial infection, we demonstrate that gentamicin dissolving microarray patches, applied to murine ears, could control K. pneumoniae infection. Mice treated with microarray patches had reduced bacterial burden in the nasal associated lymphoid tissue and lungs, in comparison to their untreated counterparts. This proof of concept study represents the first published data on the in vivo delivery of the antibiotic gentamicin, via dissolving microarray patches resulting in control of bacterial infection.

Additional file 1. Supplementary figures and tables.

Sign languages are a very interesting object of linguistic study, posing challenges not present in oral languages. One of these challenges is describing and transcribing the internal structure of the language in a way that is adequate to its characteristics but also compatible with existing linguistic practice. The phonology of sign languages is of special interest. We focus on one phonological feature: that of hand orientation. We propose an interpretation and description system that better captures underlying meaning and structure, and that is more appropriate for its formal and computational treatment. Las lenguas de signos son un objeto de estudio lingüístico de gran interés. Presentan retos y dificultades distintos a los de las lenguas orales, como describir y transcribir la estructura interna de la lengua de una manera adecuada a sus características únicas pero también compatible con la práctica lingüística actual. El caso de la fonología es especialmente interesante. Nos centramos en un rasgo fonológico concreto: la orientación de la mano. Proponemos una interpretación y un sistema de descripción que capturan mejor la semántica y la estructura subyacente, y que además permiten un tratamiento formal y computacional más adecuado. This research is partially supported by the IDiLyCo project (TIN2015-66655-R) funded by the Spanish Ministry of Economy, Industry and Competitiveness.