A peek inside: Dynamic nuclear polarization (DNP) enhances the spectroscopic sensitivity of solid‐state NMR measurements of uniformly (13C,15N)‐labeled preparations of Escherichia coli cells by more ...than an order of magnitude (see picture; MW=microwaves, ε=enhancement factor). The major molecular components in the cells can be characterized in this way.
Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted ...virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.
GNA2091 of Neisseria meningitidis is a lipoprotein of unknown function that is included in the novel 4CMenB vaccine. Here, we investigated the biological function and the subcellular localization of ...the protein. We demonstrate that GNA2091 functions in the assembly of outer membrane proteins (OMPs) because its absence resulted in the accumulation of misassembled OMPs. Cell fractionation and protease accessibility experiments showed that the protein is localized at the periplasmic side of the outer membrane. Pulldown experiments revealed that it is not stably associated with the β-barrel assembly machinery, the previously identified complex for OMP assembly. Thus, GNA2091 constitutes a novel outer membrane-based lipoprotein required for OMP assembly. Furthermore, its location at the inner side of the outer membrane indicates that protective immunity elicited by this antigen cannot be due to bactericidal or opsonic activity of antibodies.
Outer membrane protein assembly is an incompletely understood process in Gram-negative bacteria.
A Neisseria meningitidis mutant lacking the lipoprotein GNA2091 is affected in growth and accumulates unassembled outer membrane proteins.
We identified a novel component involved in outer membrane biogenesis.
Our findings contribute to the understanding of a fundamental process occurring in Gram-negative bacteria.
Neisseria meningitidis is a human pathogen. It is intensively studied for host-pathogen interactions and vaccine development. However, its favorable growth properties, genetic accessibility, and ...small genome size also make it an excellent model organism for studying fundamental biological processes, such as outer membrane biogenesis. Indeed, the first component of the assembly machinery for outer-membrane proteins, the BAM complex, was identified in N. meningitidis. Here, we describe protocols to inactivate chromosomal genes and to express genes from a well-controlled promoter on a plasmid in N. meningitidis. Together, these protocols can be used, for example, to deplete cells from essential components of the BAM complex. We also describe a simple, gel-based assay to assess the proper functioning of the BAM complex in vivo.
Previously developed whole-cell vaccines against
, the causative agent of whooping cough, appeared to be too reactogenic due to their endotoxin content. Reduction in endotoxicity can generally be ...achieved through structural modifications in the lipid A moiety of lipopolysaccharides (LPS). In this study, we found that dephosphorylation of lipid A in
through the heterologous production of the phosphatase LpxE from
did, unexpectedly, not affect Toll-like receptor 4 (TLR4)-stimulating activity. We then focused on the inner core of LPS, whose synthesis has so far not been studied in
. The
and
genes, responsible for the incorporation of a single 3-deoxy-D-
-oct-2-ulosonic acid (Kdo) residue in the inner core and its phosphorylation, respectively, appeared to be essential. However, the Kdo-bound phosphate could be replaced by a second Kdo after the heterologous production of
. This structural change in the inner core affected outer-core and lipid A structures and also bacterial physiology, as reflected in cell filamentation and a switch in virulence phase. Furthermore, the
gene responsible for the non-stoichiometric substitution of Kdo-bound phosphate with phosphoethanolamine was identified and inactivated. Interestingly, the constructed inner-core modifications affected TLR4-stimulating activity. Whereas endotoxicity studies generally focus on the lipid A moiety, our data demonstrate that structural changes in the inner core can also affect TLR4-stimulating activity.
Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental ...physiological processes. Nuclear magnetic resonance is a well-established imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution.
The Gram-negative bacterium Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but ...appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in the outer membrane (OM). Here, we investigated the possibility of reducing endotoxicity by modulating the LPS levels. The promoter of the lpxC gene, which encodes the first committed enzyme in LPS biosynthesis, was replaced by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. The IPTG was essential for growth, even when the construct was moved into a strain that should allow for the replacement of LPS in the outer leaflet of the OM with phospholipids by defective phospholipid transporter Mla and OM phospholipase A. LpxC depletion in the absence of IPTG resulted in morphological changes of the cells and in overproduction of outer-membrane vesicles (OMVs). The reduced amounts of LPS in whole-cell preparations and in isolated OMVs of LpxC-depleted cells resulted in lower activation of Toll-like receptor 4 in HEK-Blue reporter cells. We suggest that, besides lipid A engineering, also a reduction in LPS synthesis is an attractive strategy for the production of either whole-cell- or OMV-based vaccines, with reduced reactogenicity for B. pertussis and other Gram-negative bacteria.
Summary
The genome of the Gram‐negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these ...genes is induced under inorganic phosphate (Pi) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low Pi conditions and transported across the cell envelope in an Xcp‐dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin‐arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P. putida genome, of which one, named tat‐1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat‐1 genes was strongly induced by low Pi levels, indicating a function of this system in survival during Pi starvation.
Although AmpC β-lactamases can barely degrade carbapenems, if at all, they can sequester them and prevent them from reaching their targets. Thus, carbapenem resistance in Escherichia coli and other ...Enterobacteriaceae can result from AmpC production and simultaneous reduction of antibiotic influx into the periplasm by mutations in the porin genes. Here we investigated the route and genetic mechanisms of acquisition of carbapenem resistance in a clinical E. coli isolate carrying bla
on a plasmid by selecting for mutants that are resistant to increasing concentrations of meropenem. In the first step, the expression of OmpC, the only porin produced in the strain under laboratory conditions, was lost, leading to reduced susceptibility to meropenem. In the second step, the expression of the CMY-2 β-lactamase was upregulated, leading to resistance to meropenem. The loss of OmpC was due to the insertion of an IS1 element into the ompC gene or to frameshift mutations and premature stop codons in this gene. The bla
gene was found to be located on an IncIγ plasmid, and overproduction of the CMY-2 enzyme resulted from an increased plasmid copy number due to a nucleotide substitution in the inc gene. The clinical relevance of these genetic mechanisms became evident from the analysis of previously isolated carbapenem-resistant clinical isolates, which appeared to carry similar mutations.
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