Publications by authors named "Jan Tommassen"

115 Publications

Sink drains as reservoirs of VIM-2 metallo-β-lactamase-producing Pseudomonas aeruginosa in a Belgian intensive care unit: relation to patients investigated by whole genome sequencing.

J Hosp Infect 2021 Jun 7. Epub 2021 Jun 7.

Department of Microbiology and Infection Control, Vrije Universiteit Brussel (VUB), Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium.

Background: Hospital acquired infections caused by VIM encoded metallo-β-lactamase-positive Pseudomonas aeruginosa are a major problem in intensive care units (ICUs) worldwide. A previous study conducted in the UZ Brussel hospital revealed that sink drains of the ICU were a possible source of various multi-drug resistant pathogenic bacteria.

Aim: We aimed to investigate the presence and persistence of VIM P. aeruginosa in the sink drains of the four adult ICUs and their role in nosocomial infections emphasizing a sink-to-patient transmission.

Methods: Thirty-six sinks located in the ICUs of the UZ Brussel were sampled and screened for the presence of VIM P. aeruginosa in August and October 2019. Whole genome sequencing (WGS) was performed on all positive sink drain isolates together with 61 isolates from patients that were retrospectively selected (ICU patients 2019-2020, n=46; non-ICU patients 2019, n=6).

Findings: Twenty sinks were found positive for P. aeruginosa at both sampling time points. WGS revealed that the predominating environmental cluster belonged to sequence type ST111. Ten additional STs were identified. VIM-2 was detected among all ST17 (n=2) and ST111 (n=14) sink drain isolates. Based on whole-genome multilocus sequence typing analysis of all genomes, 15 clusters of highly related isolates were identified, of which seven include both sink drain and clinical isolates.

Conclusion: Our findings confirm that sink drains are a possible source of VIM-2 P. aeruginosa, probably after being contaminated with clinical waste from patients. Patients could be exposed to VIM-2 P. aeruginosa dispersed in their environment because of colonized sink drains.
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http://dx.doi.org/10.1016/j.jhin.2021.05.010DOI Listing
June 2021

Reduction of endotoxicity in Bordetella bronchiseptica by lipid A engineering: Characterization of lpxL1 and pagP mutants.

Virulence 2021 12;12(1):1452-1468

Section Molecular Microbiology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands.

Whole-cell vaccines against Gram-negative bacteria commonly display high reactogenicity caused by the endotoxic activity of lipopolysaccharide (LPS), one of the major components of the bacterial outer membrane. Underacylation of the lipid A moiety of LPS has been related with reduced endotoxicity in several Gram-negative species. Here, we evaluated whether the inactivation of two genes encoding lipid A acylases of , i.e. and , could be used for the development of less reactogenic vaccines against this pathogen for livestock and companion animals. Inactivation of resulted in the loss of the secondary palmitate chain at position 3' of lipid A, but hardly affected the potency of the LPS to activate the Toll-like receptor 4 (TLR4). Inactivation of resulted in the loss of the secondary 2-hydroxy laurate group present at position 2 of lipid A and, unexpectedly, in the additional loss of the glucosamines that decorate the phosphate groups at positions 1 and 4' and in an increase in LPS molecules carrying O-antigen. The resulting LPS showed greatly reduced potency to activate TLR4 in HEK-Blue reporter cells expressing human or mouse TLR4 as well as in porcine macrophages. Characterization of the mutant revealed many pleiotropic phenotypes, including increased resistance to SDS and rifampicin, increased susceptibility to cationic antimicrobial peptides, decreased auto-aggregation and biofilm formation, and a tendency to decreased infectivity of macrophages, which are all related to the altered LPS structure. We suggest that the mutant will be useful for the generation of safer vaccines.
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http://dx.doi.org/10.1080/21505594.2021.1929037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168481PMC
December 2021

Biofilms as Promoters of Bacterial Antibiotic Resistance and Tolerance.

Antibiotics (Basel) 2020 Dec 23;10(1). Epub 2020 Dec 23.

Unit of Microbiology and Immunology, Faculty of Veterinary, University of Zaragoza, Miguel Servet, 177, 50017 Zaragoza, Spain.

Multidrug resistant bacteria are a global threat for human and animal health. However, they are only part of the problem of antibiotic failure. Another bacterial strategy that contributes to their capacity to withstand antimicrobials is the formation of biofilms. Biofilms are associations of microorganisms embedded a self-produced extracellular matrix. They create particular environments that confer bacterial tolerance and resistance to antibiotics by different mechanisms that depend upon factors such as biofilm composition, architecture, the stage of biofilm development, and growth conditions. The biofilm structure hinders the penetration of antibiotics and may prevent the accumulation of bactericidal concentrations throughout the entire biofilm. In addition, gradients of dispersion of nutrients and oxygen within the biofilm generate different metabolic states of individual cells and favor the development of antibiotic tolerance and bacterial persistence. Furthermore, antimicrobial resistance may develop within biofilms through a variety of mechanisms. The expression of efflux pumps may be induced in various parts of the biofilm and the mutation frequency is induced, while the presence of extracellular DNA and the close contact between cells favor horizontal gene transfer. A deep understanding of the mechanisms by which biofilms cause tolerance/resistance to antibiotics helps to develop novel strategies to fight these infections.
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http://dx.doi.org/10.3390/antibiotics10010003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822488PMC
December 2020

The outer-membrane protein MafA of Neisseria meningitidis constitutes a novel protein secretion pathway specific for the fratricide protein MafB.

Virulence 2020 12;11(1):1701-1715

Section Molecular Microbiology, Department of Biology, Utrecht University , Utrecht, Netherlands.

MafB proteins are toxins secreted by spp. which are involved in interbacterial competition. Their secretion mechanism has so far not been elucidated. Each strain can produce several MafB variants. On the chromosome, the genes are localized on genomic islands also containing genes. MafA proteins have a role in virulence with reported activities in adhesion and transcytosis of pathogenic unrelated to MafB activities. In this study, we investigated the possible involvement of MafA in the transport of MafB across the outer membrane of . In wild-type strains, proteolytic fragments of MafB proteins were detected in the extracellular medium. In the absence of MafA, secretion was abrogated, and, in the case of MafB, full-length and truncated polypeptides were detected inside the cells and inside outer-membrane vesicles. MafB secretion required its cognate MafA, whereas MafB could use any MafA. Heterologous expression in showed that MafB is transported to a cell-surface-exposed, i.e. protease-accessible, location in a MafA-dependent way. MafA itself was found to be localized to the outer membrane, forming large oligomeric complexes. As homologs were found in diverse bacteria, the Maf system represents a new protein secretion system in Gram-negative bacteria.
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http://dx.doi.org/10.1080/21505594.2020.1851940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738311PMC
December 2020

Shortening the Lipid A Acyl Chains of Enables Depletion of Lipopolysaccharide Endotoxic Activity.

Vaccines (Basel) 2020 Oct 9;8(4). Epub 2020 Oct 9.

Institute for Translational Vaccinology (Intravacc), 3721 MA Bilthoven, The Netherlands.

Whooping cough, or pertussis, is an acute respiratory infectious disease caused by the Gram-negative bacterium Whole-cell vaccines, which were introduced in the fifties of the previous century and proved to be effective, showed considerable reactogenicity and were replaced by subunit vaccines around the turn of the century. However, there is a considerable increase in the number of cases in industrialized countries. A possible strategy to improve vaccine-induced protection is the development of new, non-toxic, whole-cell pertussis vaccines. The reactogenicity of whole-cell pertussis vaccines is, to a large extent, derived from the lipid A moiety of the lipopolysaccharides (LPS) of the bacteria. Here, we engineered strains with altered lipid A structures by expressing genes for the acyltransferases LpxA, LpxD, and LpxL from other bacteria resulting in altered acyl-chain length at various positions. Whole cells and extracted LPS from the strains with shorter acyl chains showed reduced or no activation of the human Toll-like receptor 4 in HEK-Blue reporter cells, whilst a longer acyl chain increased activation. Pyrogenicity studies in rabbits confirmed the in vitro assays. These findings pave the way for the development of a new generation of whole-cell pertussis vaccines with acceptable side effects.
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http://dx.doi.org/10.3390/vaccines8040594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712016PMC
October 2020

In vivo transcriptomes of Streptococcus suis reveal genes required for niche-specific adaptation and pathogenesis.

Virulence 2019 12;10(1):334-351

a Department of Infection Biology , Wageningen BioVeterinary Research (WBVR) , Lelystad , The Netherlands.

Streptococcus suis is a Gram-positive bacterium and a zoonotic pathogen residing in the nasopharynx or the gastrointestinal tract of pigs with a potential of causing life-threatening invasive disease. It is endemic in the porcine production industry worldwide, and it is also an emerging human pathogen. After invasion, the pathogen adapts to cause bacteremia and disseminates to different organs including the brain. To gain insights in this process, we infected piglets with a highly virulent strain of S. suis, and bacterial transcriptomes were obtained from blood and different organs (brain, joints, and heart) when animals had severe clinical symptoms of infection. Microarrays were used to determine the genome-wide transcriptional profile at different infection sites and during growth in standard growth medium in vitro. We observed differential expression of around 30% of the Open Reading Frames (ORFs) and infection-site specific patterns of gene expression. Genes with major changes in expression were involved in transcriptional regulation, metabolism, nutrient acquisition, stress defenses, and virulence, amongst others, and results were confirmed for a subset of selected genes using RT-qPCR. Mutants were generated in two selected genes, and the encoded proteins, i.e., NADH oxidase and MetQ, were shown to be important virulence factors in coinfection experiments and in vitro assays. The knowledge derived from this study regarding S. suis gene expression in vivo and identification of virulence factors is important for the development of novel diagnostic and therapeutic strategies to control S. suis disease.
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http://dx.doi.org/10.1080/21505594.2019.1599669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527017PMC
December 2019

Substrate specificity of the pyrophosphohydrolase LpxH determines the asymmetry of lipid A.

J Biol Chem 2019 05 29;294(20):7982-7989. Epub 2019 Mar 29.

Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of , the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3' positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in , has limited chain length specificity. However, this only partially explains the strict asymmetry of lipid A. In attempts to modulate the endotoxicity of lipid A, here we expressed the gene encoding LpxA from , which specifically attaches 3OH-C12 chains, in This expression was lethal, suggesting that one of the downstream enzymes in the lipid A biosynthesis pathway in cannot handle precursors with a 3OH-C12 chain. We considered that the UDP-diacylglucosamine pyrophosphohydrolase LpxH could be responsible for this defect as well as for the asymmetry of lipid A. Expression of meningococcal LpxH in indeed resulted in new symmetric lipid A species with 3OH-C10 or 3OH-C14 chains at both the 3 and 3' positions, as revealed by MS analysis. Furthermore, co-expression of meningococcal and resulted in viable cells that incorporated 3OH-C12 chains in lipid A. We conclude that the asymmetry of lipid A is determined by the acyl chain length specificity of LpxH.
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http://dx.doi.org/10.1074/jbc.RA118.004680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527161PMC
May 2019

Spread of Carbapenem Resistance by Transposition and Conjugation Among .

Front Microbiol 2018 5;9:2057. Epub 2018 Sep 5.

Section Molecular Microbiology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands.

The emergence of carbapenem-resistant represents a worldwide problem. To understand the carbapenem-resistance mechanisms and their spreading among strains, whole genome sequences were determined of two extensively drug-resistant strains that are endemic in Dutch hospitals. Strain Carb01 63 is of O-antigen serotype O12 and of sequence type ST111, whilst S04 90 is a serotype O11 strain of ST446. Both strains carry a gene for metallo-β-lactamase VIM-2 flanked by two genes encoding aminoglycoside acetyltransferases on a class 1 integron. The integron is located on the chromosome in strain Carb01 63 and on a plasmid in strain S04 90. The backbone of the 159-kb plasmid, designated pS04 90, is similar to a previously described plasmid, pND6-2, from . Analysis of the context of the integron showed that it is present in both strains on a ∼30-kb mosaic DNA segment composed of four different transposons that can presumably act together as a novel, active, composite transposon. Apart from the presence of a 1237-bp insertion sequence element in the composite transposon on pS04 90, these transposons show > 99% sequence identity indicating that transposition between plasmid and chromosome could have occurred only very recently. The pS04 90 plasmid could be transferred by conjugation to a susceptible strain. A second class 1 integron containing a gene for a CARB-2 β-lactamase flanked by an and an gene, encoding an aminoglycoside acetyltransferase and adenylyltransferase, respectively, was present only in strain Carb01 63. This integron is located also on a composite transposon that is inserted in an integrative and conjugative element on the chromosome. Additionally, this strain contains a frameshift mutation in the gene encoding a porin involved in the transport of carbapenems across the outer membrane. Together, the results demonstrate that integron-encoded carbapenem and carbapenicillin resistance can easily be disseminated by transposition and conjugation among strains.
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http://dx.doi.org/10.3389/fmicb.2018.02057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6133989PMC
September 2018

Biological Functions of the Secretome of .

Front Cell Infect Microbiol 2017 16;7:256. Epub 2017 Jun 16.

Department of Molecular Microbiology and Institute of Biomembranes, Utrecht UniversityUtrecht, Netherlands.

is a Gram-negative bacterial pathogen that normally resides as a commensal in the human nasopharynx but occasionally causes disease with high mortality and morbidity. To interact with its environment, it transports many proteins across the outer membrane to the bacterial cell surface and into the extracellular medium for which it deploys the common and well-characterized autotransporter, two-partner and type I secretion mechanisms, as well as a recently discovered pathway for the surface exposure of lipoproteins. The surface-exposed and secreted proteins serve roles in host-pathogen interactions, including adhesion to host cells and extracellular matrix proteins, evasion of nutritional immunity imposed by iron-binding proteins of the host, prevention of complement activation, neutralization of antimicrobial peptides, degradation of immunoglobulins, and permeabilization of epithelial layers. Furthermore, they have roles in interbacterial interactions, including the formation and dispersal of biofilms and the suppression of the growth of bacteria competing for the same niche. Here, we will review the protein secretion systems of and focus on the functions of the secreted proteins.
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http://dx.doi.org/10.3389/fcimb.2017.00256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472700PMC
February 2018

Interstrain Cooperation in Meningococcal Biofilms: Role of Autotransporters NalP and AutA.

Front Microbiol 2017 22;8:434. Epub 2017 Mar 22.

Section Molecular Microbiology, Department of Biology, Utrecht University Utrecht, Netherlands.

() and () are commensal bacteria that live in the human nasopharynx, where they form microcolonies. In contrast to occasionally causes blood and/or meningitis infection with often fatal consequences. Here, we studied interactions between neisserial strains during biofilm formation. Fluorescent strains were engineered and analyzed for growth in single- and dual-strain biofilms with confocal laser-scanning microscopy. Different strains of diverse species formed microcolonies of different sizes and morphologies. Pair-wise combinations of two invasive strains and one carrier isolate showed that these strains can coexist in spite of the fact that they produce toxins to combat congeners. This lack of competition was even observed when the biofilms were formed under nutrient limitation and can be explained by the observation that the separate microcolonies within mixed biofilms are mostly lineage specific. However, these microcolonies showed different levels of interaction. The coexistence of two strains was also observed in mixed biofilms of and strains. Inactivation of the autotransporter NalP, which prevents the release of the heparin-binding antigen NHBA and the α-peptide of IgA protease from the cell surface, and/or the production of autotransporter AutA increased interactions between microcolonies, as evidenced by close contacts between microcolonies on the substratum. Qualitative and quantitative analysis revealed an altered spatial distribution of each strain in mixed biofilms with consequences for the biomass, biofilm architecture and bacterial viability depending on the synthesis of NalP and AutA, the expression of which is prone to phase variation. Being in a consortium resulted in some cases in commensalism and cooperative behavior, which promoted attachment to the substratum or increased survival, possibly as result of the shared use of the biofilm matrix. We hypothesize that strains can cooperate during host colonization, but, possibly, the different capacities of the microcolonies of each strain to resist the host's defenses limits the long-term coexistence of strains in the host.
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http://dx.doi.org/10.3389/fmicb.2017.00434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360712PMC
March 2017

Expression of the Gene for Autotransporter AutB of Affects Biofilm Formation and Epithelial Transmigration.

Front Cell Infect Microbiol 2016 22;6:162. Epub 2016 Nov 22.

Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University Utrecht, Netherlands.

is a Gram-negative bacterium that resides as a commensal in the upper respiratory tract of humans, but occasionally, it invades the host and causes sepsis and/or meningitis. The bacterium can produce eight autotransporters, seven of which have been studied to some detail. The remaining one, AutB, has not been characterized yet. Here, we show that the gene is broadly distributed among pathogenic spp. The gene is intact in most meningococcal strains. However, its expression is prone to phase variation due to slipped-strand mispairing at AAGC repeats located within the DNA encoding the signal sequence and is switched off in the vast majority of these strains. Moreover, various genetic disruptions prevent expression in most of the strains in which the gene is in phase indicating a strong selection against AutB synthesis. We observed that is expressed in two of the strains examined and that AutB is secreted and exposed at the cell surface. Functionality assays revealed that AutB synthesis promotes biofilm formation and delays the passage of epithelial cell layers in . We hypothesize that this autotransporter is produced during the colonization process only in specific niches to facilitate microcolony formation, but its synthesis is switched off probably to evade the immune system and facilitate human tissue invasion.
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http://dx.doi.org/10.3389/fcimb.2016.00162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5118866PMC
August 2017

Acquisition of Carbapenem Resistance by Plasmid-Encoded-AmpC-Expressing Escherichia coli.

Antimicrob Agents Chemother 2017 01 27;61(1). Epub 2016 Dec 27.

Department of Molecular Microbiology, Institute of Biomembranes, Utrecht University, Utrecht, Netherlands

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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http://dx.doi.org/10.1128/AAC.01413-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192137PMC
January 2017

Meningococcal Biofilm Formation: Let's Stick Together.

Trends Microbiol 2017 02 3;25(2):113-124. Epub 2016 Oct 3.

Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Extracellular DNA (eDNA) is an essential constituent of the extracellular matrix of biofilms of many microorganisms. In spite of many studies, it has long remained unclear how exactly eDNA exerts its role in biofilm formation. Here, we describe recent advances that have been made in understanding biofilm formation in the human pathogen Neisseria meningitidis. Several cell-surface-exposed proteins have been identified that bind DNA and other negatively charged polymers, such as heparin, by electrostatic interactions. By virtue of these proteins, eDNA can act as an adhesive that binds the bacteria together. We provide examples that indicate that the mechanism of binding eDNA via DNA/heparin-binding proteins is a conserved feature in biofilms of many different microorganisms, including fungi.
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http://dx.doi.org/10.1016/j.tim.2016.09.005DOI Listing
February 2017

Experimental Methods for Studying the BAM Complex in Neisseria meningitidis.

Methods Mol Biol 2015 ;1329:33-49

Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, Utrecht, 3584 CH, The Netherlands.

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.
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http://dx.doi.org/10.1007/978-1-4939-2871-2_3DOI Listing
July 2016

Fratricide activity of MafB protein of N. meningitidis strain B16B6.

BMC Microbiol 2015 Aug 5;15:156. Epub 2015 Aug 5.

Department of Molecular Microbiology, Utrecht University, Padualaan 8, 3584, CH, Utrecht, The Netherlands.

Background: Neisseria meningitidis is an inhabitant of the mucosal surfaces of the human nasopharynx. We recently demonstrated that the secreted meningococcal Two-partner secretion protein A (TpsA) is involved in interbacterial competition. The C-terminal end of the large TpsA protein contains a small toxic domain that inhibits the growth of target bacteria. The producing cells are protected from this toxic activity by a small immunity protein that is encoded by the gene immediately downstream of the tpsA gene. Further downstream on the chromosome, a repertoire of toxic modules, designated tpsC cassettes, is encoded that could replace the toxic module of TpsA by recombination. Each tpsC cassette is associated with a gene encoding a cognate immunity protein.

Results: Blast searchers using the toxic domains of TpsA and TpsC proteins as queries identified homologies with the C-terminal part of neisserial MafB proteins, which, for the rest, showed no sequence similarity to TpsA proteins. On the chromosome, mafB genes are part of genomic islands, which include cassettes for additional toxic modules as well as genes putatively encoding immunity proteins. We demonstrate that a MafB protein of strain B16B6 inhibits the growth of a strain that does not produce the corresponding immunity protein. Assays in E. coli confirmed that the C-terminal region of MafB is responsible for toxicity, which is inhibited by the cognate immunity protein. Pull-down assays revealed direct interaction between MafB toxic domains and the cognate immunity proteins.

Conclusions: The meningococcal MafB proteins are novel toxic proteins involved in interbacterial competition.
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http://dx.doi.org/10.1186/s12866-015-0493-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524018PMC
August 2015

Unearthing the genomes of plant-beneficial Pseudomonas model strains WCS358, WCS374 and WCS417.

BMC Genomics 2015 Jul 22;16:539. Epub 2015 Jul 22.

Plant-Microbe Interactions, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

Background: Plant growth-promoting rhizobacteria (PGPR) can protect plants against pathogenic microbes through a diversity of mechanisms including competition for nutrients, production of antibiotics, and stimulation of the host immune system, a phenomenon called induced systemic resistance (ISR). In the past 30 years, the Pseudomonas spp. PGPR strains WCS358, WCS374 and WCS417 of the Willie Commelin Scholten (WCS) collection have been studied in detail in pioneering papers on the molecular basis of PGPR-mediated ISR and mechanisms of biological control of soil-borne pathogens via siderophore-mediated competition for iron.

Results: The genomes of the model WCS PGPR strains were sequenced and analyzed to unearth genetic cues related to biological questions that surfaced during the past 30 years of functional studies on these plant-beneficial microbes. Whole genome comparisons revealed important novel insights into iron acquisition strategies with consequences for both bacterial ecology and plant protection, specifics of bacterial determinants involved in plant-PGPR recognition, and diversity of protein secretion systems involved in microbe-microbe and microbe-plant communication. Furthermore, multi-locus sequence alignment and whole genome comparison revealed the taxonomic position of the WCS model strains within the Pseudomonas genus. Despite the enormous diversity of Pseudomonas spp. in soils, several plant-associated Pseudomonas spp. strains that have been isolated from different hosts at different geographic regions appear to be nearly isogenic to WCS358, WCS374, or WCS417. Interestingly, all these WCS look-a-likes have been selected because of their plant protective or plant growth-promoting properties.

Conclusions: The genome sequences of the model WCS strains revealed that they can be considered representatives of universally-present plant-beneficial Pseudomonas spp. With their well-characterized functions in the promotion of plant growth and health, the fully sequenced genomes of the WCS strains provide a genetic framework that allows for detailed analysis of the biological mechanisms of the plant-beneficial traits of these PGPR. Considering the increasing focus on the role of the root microbiome in plant health, functional genomics of the WCS strains will enhance our understanding of the diversity of functions of the root microbiome.
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http://dx.doi.org/10.1186/s12864-015-1632-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4509608PMC
July 2015

Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells.

PLoS Pathog 2015 Jun 17;11(6):e1004946. Epub 2015 Jun 17.

Department of Pulmonology, Leiden University Medical Centre, Leiden, the Netherlands.

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.
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http://dx.doi.org/10.1371/journal.ppat.1004946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4471080PMC
June 2015

Transport of lipopolysaccharide to the Gram-negative bacterial cell surface.

FEMS Microbiol Rev 2015 Nov 1;39(6):985-1002. Epub 2015 Jun 1.

Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, the Netherlands

Lipopolysaccharides (LPS) are major lipidic components of the outer membrane of most Gram-negative bacteria. They form a permeability barrier that protects these bacteria from harmful compounds in the environment. In addition, they are important signaling molecules for the innate immune system. The mechanism of transport of these molecules to the bacterial cell surface has remained enigmatic for a long time. However, intense research during the last decade, particularly in Escherichia coli and Neisseria meningitidis, has led to the identification of the machinery that mediates LPS transport. In this review, we summarize the current knowledge of the LPS transport machinery and provide an overview of the distribution of the components of this machinery among diverse bacteria, even organisms that don't produce LPS. We also discuss the current insights in the regulation of LPS biosynthesis.
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http://dx.doi.org/10.1093/femsre/fuv026DOI Listing
November 2015

Reprint of “Inhibition of biofilm formation by Camelid single-domainantibodies against the flagellum of Pseudomonas aeruginosa”.

J Biotechnol 2014 Dec;191:131-8

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in patients with compromised host defense mechanisms, including burn wound victims. In addition to its intrinsic resistance against most antibiotics, P. aeruginosa has the ability to form biofilms adhering to biotic or abiotic surfaces. These factors make treatment of P. aeruginosa infections complicated and demand new therapies and drugs. The flagellum of P. aeruginosa plays an important role in cell–cell and cell–surface interactions during the first stage of biofilm formation. In this study, we describe the selection of monoclonal anti-flagellin single-domain antibodies (VHHs) derived from the Camelid heavy-chain antibody repertoire of a llama immunized with P. aeruginosa antigens. The anti-flagellin VHHs could be produced efficiently in Saccharomyces cerevisiae, and surface plasmon resonance experiments demonstrated that they have apparent affinities in the nanomolar range. Functional screens showed that the anti-flagellin VHHs are capable of inhibiting P. aeruginosa from swimming and that they prevent biofilm formation in an in vitro assay. These data open doors for the development of novel methods for the prevention of P. aeruginosa-related infections.
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http://dx.doi.org/10.1016/j.jbiotec.2014.09.022DOI Listing
December 2014

Increased expression levels of chromosomal AmpC β-lactamase in clinical Escherichia coli isolates and their effect on susceptibility to extended-spectrum cephalosporins.

Microb Drug Resist 2015 Feb 4;21(1):7-16. Epub 2014 Sep 4.

1 Department of Medical Microbiology, Leiden University Medical Center , Leiden, The Netherlands .

Forty-nine clinical Escherichia coli isolates, both extended-spectrum β-lactamase (ESBL) negative and ESBL positive, were studied to investigate whether increased AmpC expression is a mechanism involved in cefoxitin resistance and if this influences the third-generation cephalosporin activity. Nine of 33 (27.2%) cefoxitin-resistant (minimum inhibitory concentration [MIC] >8 mg/L) isolates showed hyperproduction of chromosomal AmpC (c-AmpC) based on (1) at least two positive tests using AmpC inhibitors, (2) mutations in the promoter/attenuator regions, and (3) a 6.1- to 163-fold increase in c-ampC expression by quantitative reverse transcription-polymerase chain reaction. In ESBL-negative isolates, MICs of ceftazidime and cefotaxime were mostly above the wild-type (WT) level, but below the S/I breakpoint (EUCAST guideline), except for one isolate with MICs of 4 mg/L. No plasmid-mediated AmpCs were found. Periplasmic extracts of nine c-AmpC hyperproducers were preincubated with or without cefuroxime or ceftazidime and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cefuroxime and ceftazidime were stable to hydrolysis but acted as inhibitors of the enzyme. None of these isolates showed loss of porins. Thus, cefoxitin resistance has low specificity for detecting upregulated c-AmpC production. c-AmpC hyperproducing E. coli is mostly still susceptible to third-generation cephalosporins but less than WT E. coli. Surveillance of cefoxitin-resistant E. coli to monitor developments in the activity of third-generation cephalosporins against c-AmpC hyperproducers is warranted.
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http://dx.doi.org/10.1089/mdr.2014.0108DOI Listing
February 2015

Variable processing of the IgA protease autotransporter at the cell surface of Neisseria meningitidis.

Microbiology (Reading) 2014 Nov 26;160(Pt 11):2421-2431. Epub 2014 Aug 26.

Department of Molecular Microbiology, Institute of Molecular Cell Biology, VU University, 1081 HV Amsterdam, The Netherlands.

As with all classical monomeric autotransporters, IgA protease of Neisseria meningitidis is a modular protein consisting of an N-terminal signal sequence, a passenger domain and a C-terminal translocator domain (TD) that assists in the secretion of the passenger domain across the outer membrane. The passenger of IgA protease consists of three separate domains: the protease domain, the γ-peptide and the α-peptide that contains nuclear localization signals (NLSs). The protease domain is released into the extracellular milieu either via autocatalytic processing or via cleavage by another autotransporter, NalP, expression of which is phase-variable. NalP-mediated cleavage results in the release of a passenger that includes the α- and γ-peptides. Here, we studied the fate of the α-peptide when NalP was not expressed and observed strain-dependent differences. In meningococcal strains where the α-peptide contained a single NLS, the α-peptide remained covalently attached to the TD and was detected at the cell surface. In other strains, the α-peptide contained four NLSs and was separated from the TD by an IgA protease autoproteolytic cleavage site. In many of those cases, the α-peptide was found non-covalently associated with the cells as a separate polypeptide. The cell surface association of the α-peptides may be relevant physiologically. We report a novel function for the α-peptide, i.e. the binding of heparin - an immune-modulatory molecule that in the host is found in the extracellular matrix and connected to cell surfaces.
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http://dx.doi.org/10.1099/mic.0.082511-0DOI Listing
November 2014

The meningococcal autotransporter AutA is implicated in autoaggregation and biofilm formation.

Environ Microbiol 2015 Apr 9;17(4):1321-37. Epub 2014 Sep 9.

Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Paudalaan 8, Utrecht, 3584 CH, The Netherlands.

Autotransporters (ATs) are proteins secreted by Gram-negative bacteria that often play a role in virulence. Eight different ATs have been identified in Neisseria meningitidis, but only six of them have been characterized. AutA is one of the remaining ATs. Its expression remains controversial. Here, we show that the autA gene is present in many neisserial species, but its expression is often disrupted by various genetic features; however, it is expressed in certain strains of N. meningitidis. By sequencing the autA gene in large panels of disease isolates and Western blot analysis, we demonstrated that AutA expression is prone to phase variation at AAGC nucleotide repeats located within the DNA encoding the signal sequence. AutA is not secreted into the extracellular medium, but remains associated with the bacterial cell surface. We further demonstrate that AutA expression induces autoaggregation in a process that, dependent on the particular strain, may require extracellular DNA (eDNA). This property influences the organization of bacterial communities like lattices and biofilms. In vitro assays evidenced that AutA is a self-associating AT that binds DNA. We suggest that AutA-mediated autoaggregation might be particularly important for colonization and persistence of the pathogen in the nasopharynx of the host.
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http://dx.doi.org/10.1111/1462-2920.12581DOI Listing
April 2015

Inhibition of biofilm formation by Camelid single-domain antibodies against the flagellum of Pseudomonas aeruginosa.

J Biotechnol 2014 Sep 2;186:66-73. Epub 2014 Jul 2.

Department of Molecular Microbiology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in patients with compromised host defense mechanisms, including burn wound victims. In addition to its intrinsic resistance against most antibiotics, P. aeruginosa has the ability to form biofilms adhering to biotic or abiotic surfaces. These factors make treatment of P. aeruginosa infections complicated and demand new therapies and drugs. The flagellum of P. aeruginosa plays an important role in cell-cell and cell-surface interactions during the first stage of biofilm formation. In this study, we describe the selection of monoclonal anti-flagellin single-domain antibodies (VHHs) derived from the Camelid heavy-chain antibody repertoire of a llama immunized with P. aeruginosa antigens. The anti-flagellin VHHs could be produced efficiently in Saccharomyces cerevisiae, and surface plasmon resonance experiments demonstrated that they have apparent affinities in the nanomolar range. Functional screens showed that the anti-flagellin VHHs are capable of inhibiting P. aeruginosa from swimming and that they prevent biofilm formation in an in vitro assay. These data open doors for the development of novel methods for the prevention of P. aeruginosa-related infections.
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http://dx.doi.org/10.1016/j.jbiotec.2014.06.029DOI Listing
September 2014

Involvement of Neisseria meningitidis lipoprotein GNA2091 in the assembly of a subset of outer membrane proteins.

J Biol Chem 2014 May 22;289(22):15602-10. Epub 2014 Apr 22.

From the Department of Molecular Microbiology, Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1074/jbc.M113.539510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140915PMC
May 2014

Solid-state NMR studies of full-length BamA in lipid bilayers suggest limited overall POTRA mobility.

J Mol Biol 2014 May 13;426(9):2009-21. Epub 2014 Feb 13.

NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Electronic address:

The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA (POlypeptide-TRansport-Associated) domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments. We detect POTRA domains in ssNMR experiments probing rigid protein segments in our preparations. These results suggest that the periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3. We show that this behavior holds at lower protein concentrations and elevated temperatures. Chemical shift variations observed after reconstitution in lipids with different chain lengths and saturation levels are compatible with conformational plasticity of BamA's transmembrane domain. Electron microscopy of the ssNMR samples shows that BamA can cause local disruptions of the lipid bilayer in proteoliposomes. The observed interplay between protein-protein and protein-lipid interactions may be critical for BamA-mediated insertion of substrates into the outer membrane.
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http://dx.doi.org/10.1016/j.jmb.2014.02.007DOI Listing
May 2014

Species-specificity of the BamA component of the bacterial outer membrane protein-assembly machinery.

PLoS One 2013 20;8(12):e85799. Epub 2013 Dec 20.

Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.

The BamA protein is the key component of the Bam complex, the assembly machinery for outer membrane proteins (OMP) in gram-negative bacteria. We previously demonstrated that BamA recognizes its OMP substrates in a species-specific manner in vitro. In this work, we further studied species specificity in vivo by testing the functioning of BamA homologs of the proteobacteria Neisseria meningitidis, Neisseria gonorrhoeae, Bordetella pertussis, Burkholderia mallei, and Escherichia coli in E. coli and in N. meningitidis. We found that no BamA functioned in another species than the authentic one, except for N. gonorrhoeae BamA, which fully complemented a N. meningitidis bamA mutant. E. coli BamA was not assembled into the N. meningitidis outer membrane. In contrast, the N. meningitidis BamA protein was assembled into the outer membrane of E. coli to a significant extent and also associated with BamD, an essential accessory lipoprotein of the Bam complex.Various chimeras comprising swapped N-terminal periplasmic and C-terminal membrane-embedded domains of N. meningitidis and E. coli BamA proteins were also not functional in either host, although some of them were inserted in the OM suggesting that the two domains of BamA need to be compatible in order to function. Furthermore, conformational analysis of chimeric proteins provided evidence for a 16-stranded β-barrel conformation of the membrane-embedded domain of BamA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085799PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3869937PMC
July 2014

Ght protein of Neisseria meningitidis is involved in the regulation of lipopolysaccharide biosynthesis.

J Bacteriol 2014 Feb 2;196(4):780-9. Epub 2013 Dec 2.

Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Utrecht, Netherlands.

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and is responsible for the barrier function of this membrane. A ght mutant of Neisseria meningitidis that showed increased sensitivity to hydrophobic toxic compounds, suggesting a breach in this permeability barrier, was previously described. Here, we assessed whether this phenotype was possibly caused by a defect in LPS transport or synthesis. The total amount of LPS appeared to be drastically reduced in a ght mutant, but the residual LPS was still detected at the cell surface, suggesting that LPS transport was not impaired. The ght mutant was rapidly overgrown by pseudorevertants that produced normal levels of LPS. Genetic analysis of these pseudorevertants revealed that the lpxC gene, which encodes a key enzyme in LPS synthesis, was fused to the promoter of the upstream-located pilE gene, resulting in severe lpxC overexpression. Analysis of phoA and lacZ gene fusions indicated that Ght is an inner membrane protein with an N-terminal membrane anchor and its bulk located in the cytoplasm, where it could potentially interact with LpxC. Cell fractionation experiments indeed indicated that Ght tethers LpxC to the membrane. We suggest that Ght regulates LPS biosynthesis by affecting the activity of LpxC. Possibly, this mechanism acts in the previously observed feedback inhibition of LPS synthesis that occurs when LPS transport is hampered.
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http://dx.doi.org/10.1128/JB.00943-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911185PMC
February 2014

Zinc piracy as a mechanism of Neisseria meningitidis for evasion of nutritional immunity.

PLoS Pathog 2013 Oct 31;9(10):e1003733. Epub 2013 Oct 31.

Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Utrecht, Netherlands.

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as "nutritional immunity." The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.
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http://dx.doi.org/10.1371/journal.ppat.1003733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814407PMC
October 2013

Domain exchange at the 3' end of the gene encoding the fratricide meningococcal two-partner secretion protein A.

BMC Genomics 2013 Sep 14;14:622. Epub 2013 Sep 14.

Department of Molecular Microbiology, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.

Background: Two-partner secretion systems in Gram-negative bacteria consist of an outer membrane protein TpsB that mediates the secretion of a cognate TpsA protein into the extracellular milieu. TpsA proteins have diverse, often virulence-related functions, and some of them inhibit the growth of related bacteria. In Neisseria meningitidis, several functions have been attributed to the TpsA proteins. Downstream of the tpsB and tpsA genes, several shorter tpsA-related gene cassettes, called tpsC, are located interspersed with intervening open-reading frames (IORFs). It has been suggested that the tpsC cassettes may recombine with the tpsA gene as a mechanism of antigenic variation. Here, we investigated (i) whether TpsA of N. meningitidis also has growth-inhibitory properties, (ii) whether tpsC cassettes recombine with the tpsA gene, and (iii) what the consequences of such recombination events might be.

Results: We demonstrate that meningococcal TpsA has growth-inhibitory properties and that the IORF located immediately downstream of tpsA confers immunity to the producing strain. Although bioinformatics analysis suggests that recombination between tpsC cassettes and tpsA occurs, detailed analysis of the tpsA gene in a large collection of disease isolates of three clonal complexes revealed that the frequency is very low and cannot be a mechanism of antigenic variation. However, recombination affected growth inhibition. In vitro experiments revealed that recombination can be mediated through acquirement of tpsC cassettes from the environment and it identified the regions involved in the recombination.

Conclusions: Meningococcal TpsA has growth-inhibitory properties. Recombination between tpsA and tpsC cassettes occurs in vivo but is rare and has consequences for growth inhibition. A recombination model is proposed and we propose that the main goal of recombination is the collection of new IORFs for protection against a variety of TpsA proteins.
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http://dx.doi.org/10.1186/1471-2164-14-622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3848433PMC
September 2013

Antibiotic trapping by plasmid-encoded CMY-2 β-lactamase combined with reduced outer membrane permeability as a mechanism of carbapenem resistance in Escherichia coli.

Antimicrob Agents Chemother 2013 Aug 3;57(8):3941-9. Epub 2013 Jun 3.

Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands.

A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CR E. coli lacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CR E. coli possessed bla(CTX-M-15) and bla(OXA-1). The CR E. coli strain additionally harbored bla(CMY-2) and demonstrated a >15-fold increase in β-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation either in vitro or in vivo with meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into various E. coli strains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated β-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the β-lactamase. This study, therefore, provides evidence that the mechanism of "trapping" by CMY-2 β-lactamase plays a role in carbapenem resistance.
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http://dx.doi.org/10.1128/AAC.02459-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719783PMC
August 2013
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