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

BACKGROUND: Hospital-acquired infections caused by VIM-encoded metallo-ß-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 multidrug-resistant pathogenic bacteria. AIM: 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 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 who 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 multi-locus sequence typing analysis of all genomes, 15 clusters of highly related isolates were identified, of which seven included 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.

Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant.

In the pathogen Neisseria meningitidis, a completely lipopolysaccharide (LPS)-deficient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP-GlcNAc acyltransferase required for the first step of lipid A biosynthesis. To study how outer membrane structure and biogenesis are affected by the absence of this normally major component, inner and outer membranes were separated and their composition analysed. The expression and assembly of integral outer membrane proteins appeared largely unaffected. However, the expression of iron limitation-inducible, cell surface-exposed lipoproteins was greatly reduced. Major changes were seen in the phospholipid composition, with a shift towards phosphatidylethanolamine and phosphatidylglycerol species containing mostly shorter chain, saturated fatty acids, one of which was unique to the LPS-deficient outer membrane. The presence of the capsular polysaccharide turned out to be essential for viability without LPS, as demonstrated by using a strain in which LPS biosynthesis could be switched on or off through a tac promoter-controlled lpxA gene. Taken together, these results can help to explain why meningococci have the unique ability to survive without LPS.

In vivo expression of Neisseria meningitidis proteins homologous to the Haemophilus influenzae Hap and Hia autotransporters.

The genome sequences of Neisseria meningitidis serogroup B strain MC58 and serogroup A strain Z2491 were systematically searched for open reading frames (ORFs) encoding autotransporters. Eight ORFs were identified, six of which were present in both genomes, whereas two were specific for MC58. Among the identified ORFs was the gene encoding the known autotransporter IgA1 protease. The deduced amino acid sequences of the other identified ORFs were homologous to known autotransporters and found to contain an N-terminal signal sequence and a C-terminal domain that could constitute a beta-barrel in the outer membrane. The ORFs NMB1985 and NMB0992, encoding homologs of the Hap (for Haemophilus adhesion and penetration protein) and Hia (for Haemophilus influenzae adherence protein) autotransporters of H. influenzae, were cloned from serogroup B strain H44/76 and expressed in Escherichia coli. Western blots revealed that all sera of patients (n=14) and healthy carriers (n=3) tested contained antibodies against at least one of the recombinant proteins. These results indicate that both genes are widely distributed among N. meningitidis isolates and expressed during colonization and infection.

Expression of the pho regulon interferes with induction of the uhpT gene in Escherichia coli K-12.

The uptake of hexose 6-phosphates in Escherichia coli is mediated by the transporter UhpT, the synthesis of which is induced by the presence of glucose 6-phosphate (glucose 6-P) in the medium. Since this protein functions as an anion exchanger, it is generally assumed to be geared for the use of sugar phosphates as a carbon source. However, the question was unresolved whether this transporter can also provide the cells with glucose 6-P as a phosphate source. It is demonstrated in this work that UhpT-mediated glucose 6-P uptake does not allow the cells to grow on glucose 6-P as phosphate source. Hence, the expression of UhpT under phosphate limitation would not be particularly advantageous and some form of interaction between the uhp system and the Pi-limitation-inducible pho regulon, the products of which are involved in phosphate acquisition, may be anticipated. Indeed, the use of an uhpT-lacZ fusion revealed that much higher concentrations of the inducer glucose 6-P were required to elevate uhpT transcription when the pho regulon was expressed. This interference was the result of degradation of glucose 6-P by one of the products of the pho regulon, the periplasmic enzyme alkaline phosphatase. The specific form of interaction between the Pho and Uhp systems is designated inducer degradation.

Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein.

The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.

The phosphate-binding protein of Escherichia coli is not essential for P(i)-regulated expression of the pho regulon.

Disruption of pstS encoding the P(i)-binding protein in Escherichia coli generally leads to the constitutive expression of the pho regulon. We demonstrate that P(i)-controlled expression is restored when the activity of the P(i) transporter PitA or PitB is increased. Apparently, PstS is not an essential component of the signal transduction pathway.

Autoamplification of a two-component regulatory system results in "learning" behavior.

We have tested the hypothesis that the autoamplification of two-component regulatory systems results in "learning" behavior, i.e., that bacteria respond faster or more extensively to a signal when a similar signal has been perceived in the past. Indeed, the induction of alkaline phosphatase activity upon phosphate limitation was faster if the cultures had been limited for phosphate previously, and this faster response correlated with the autoamplification of the cognate two-component system.

Activation by gene amplification of pitB, encoding a third phosphate transporter of Escherichia coli K-12.

Two systems for the uptake of inorganic phosphate (P(i)) in Escherichia coli, PitA and Pst, have been described. A revertant of a pitA pstS double mutant that could grow on P(i) was isolated. We demonstrate that the expression of a new P(i) transporter, PitB, is activated in this strain by a gene amplification event.

Maturation of Pseudomonas aeruginosa elastase. Formation of the disulfide bonds.

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. After propeptide-mediated folding in the periplasm, the proenzyme is autoproteolytically processed, prior to translocation of both the mature enzyme and the propeptide across the outer membrane. The formation of the two disulfide bonds present in the mature enzyme was examined by studying the expression of the wild-type enzyme and of alanine for cysteine mutant derivatives in the authentic host and in dsb mutants of Escherichia coli. It appeared that the two disulfide bonds are formed successively. First, DsbA catalyzes the formation of the disulfide bond between Cys-270 and Cys-297 within the proenzyme. This step is essential for the subsequent autoproteolytic processing to occur. The second disulfide bond between Cys-30 and Cys-57 is formed more slowly and appears to be formed after processing of the proenzyme, and its formation is catalyzed by DsbA as well. This second disulfide bond appeared to be required for the full proteolytic activity of the enzyme and contributes to its stability.

Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition.

Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

Phase-variable expression of an operon encoding extracellular alkaline protease, a serine protease homolog, and lipase in Pseudomonas brassicacearum.

The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation.

Identification of phospholipids as new components that assist in the in vitro trimerization of a bacterial pore protein.

The in vitro trimerization of folded monomers of the bacterial pore protein PhoE, into its native-like, heat- and SDS-stable form requires incubations with isolated cell envelopes and Triton X-100. The possibility that membranes could be isolated that are enriched in assembly factors required for assembly of the pore protein was now investigated. Fractionation of total cell envelopes of Escherichia coli via various techniques indeed revealed the existence of membrane fractions with different capacities to support assembly in vitro. Fractions containing mainly inner membrane vesicles supported the formation of trimers that were associated with these membrane vesicles. However, only a proportion of these trimers were heat- and SDS-stable and these were formed with slow kinetics. In contrast, fractions containing mainly outer membrane vesicles supported formation of high amounts of heat-stable trimers with fast kinetics. We identified phospholipids as active assembly components in these membranes that support trimerization of folded monomers in a process with similar characteristics as observed with inner membrane vesicles. Furthermore, phospholipids strongly stimulate the kinetics of trimerization and increase the final yield of heat-stable trimers in the context of outer membranes. We propose that lipopolysaccharides stabilize the assembly competent state of folded monomers as a lipochaperone. Phospholipids are involved in converting the folded monomer into new assembly competent intermediate with a short half-life that will form heat-stable trimers most efficiently in the context of outer membrane vesicles. These results provide biochemical evidence for the involvement of different lipidic components at distinct stages of the porin assembly process.

Protein secretion mechanisms in Gram-negative bacteria.

Gram-negative bacteria have developed a variety of secretion pathways to secrete toxins and enzymes into the extracellular medium. These pathways are very different with respect to their functional mechanism and complexity, and each system has its own advantages and limitations, regarding the number, size, folding state and fate of their substrates. Pseudomonas aeruginosa secretes many different proteins into the extracellular medium, using at least four secretion pathways. Most of the exoproteins are secreted via the type II system, composed of the 12 Xcp proteins. The only outer membrane protein of the system, XcpQ, belongs to a large family of proteins, designated secretins, which participate in a variety of different transport processes. Other Xcp proteins, XcpT-X, show homology to the subunits of the retractile type IV pili. Further analogies between the type II system and the assembly of retractile pili suggest a mechanism for type II secretion, in which a pilus-like structure, composed of XcpT-X, facilitates the transport of exoproteins through the channel formed by the secretin XcpQ.

Novel topological features of FhaC, the outer membrane transporter involved in the secretion of the Bordetella pertussis filamentous hemagglutinin.

Many pathogenic Gram-negative bacteria secrete virulence factors across the cell envelope into the extracellular milieu. The secretion of filamentous hemagglutinin (FHA) by Bordetella pertussis depends on the pore-forming outer membrane protein FhaC, which belongs to a growing family of protein transporters. Protein alignment and secondary structure predictions indicated that FhaC is likely to be a beta-barrel protein with an odd number of transmembrane beta-strands connected by large surface loops and short periplasmic turns. The membrane topology of FhaC was investigated by random insertion of the c-Myc epitope and the tobacco etch virus protease-specific cleavage sequence. FhaC was fairly permissive to short linker insertions. Furthermore, FhaC appeared to undergo conformational changes upon FHA secretion. Surface detection of the inserted sequences indicated that several predicted loops in the C-terminal moiety as well as the N terminus of the protein are exposed. However, a large surface-predicted region in the N-terminal moiety of FhaC was inaccessible from the surface. In addition, the activity and the stability of the protein were affected by insertions in that region, indicating that it may have important structural and/or functional roles. The surface exposure of the N terminus and the presence of an odd number of beta-strands are novel features for beta-barrel outer membrane proteins.

Role of the lipase-specific foldase of Burkholderia glumae as a steric chaperone.

Most lipases of Gram-negative bacteria require a lipase-specific foldase (Lif) in order to fold in the periplasm into their active, protease-resistant conformation prior to their secretion. The periplasmic domain of the Lif (amino acids 44-353) of Burkholderia glumae was purified as a His-tagged protein, and its function in the folding of lipase was studied in vitro. Refolding of the denatured lipase into its active conformation was dependent on the presence of the Lif. Circular dichroism revealed that the lipase refolded in the absence of Lif into a form with a native-like conformation, which was more stable against heat-induced denaturation than the native form, but was enzymatically inactive. This form of the protein could be activated by adding Lif after several hours, which demonstrates that the function of this chaperone is to help lipase to overcome an energetic barrier in the productive folding pathway rather than to prevent it from entering a non-productive pathway. The Lif was shown to interact with the native lipase in protease-protection experiments as well as by affinity chromatography, consistent with a role of the Lif late in the folding process. These results demonstrate that the Lif functions in a way analogous to the propeptides of many bacterial proteases and indicate that the amino acid sequence of the lipase does not contain all the information required for the protein to adopt its three-dimensional structure.

The assembly pathway of outer membrane protein PhoE of Escherichia coli.

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.

Biochemical and biophysical characterization of in vitro folded outer membrane porin PorA of Neisseria meningitidis.

Two subtypes of the outer membrane porin PorA of Neisseria meningitidis, P1.6 and P1.7,16, were folded in vitro after overexpression in, and isolation from Escherichia coli. The PorA porins could be folded efficiently by quick dilution in an appropriate buffer containing the detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate. Although the two PorA porins are highly homologous, they required different acidities for optimal folding, that is, a pH above the pI was needed for efficient folding. Furthermore, whereas trimers of PorA P1.7,16 were almost completely stable in 2% sodium dodecyl sulphate (SDS), those of P1.6 dissociated in the presence of SDS. The higher electrophoretic mobility of the in vitro folded porins could be explained by the stable association of the RmpM protein to the porins in vivo. This association of RmpM contributes to the stability of the porins. The P1.6 pores were moderately cation-selective and displayed a single-channel conductance of 2.8 nS in 1 M KCl. The PorA P1.6 pores, but not the PorA P1.7,16 pores, showed an unusual non-linear dependence of the single-channel conductance on the salt concentration of the subphase. We hypothesize that a cluster of three negatively charged residues in L5 of P1.6 is responsible for the higher conductance at low salt concentrations.

Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa.

One of the major proteins secreted by Pseudomonas aeruginosa is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263-270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of Streptomyces olivaceoviridis and CBP21 of Serratia marcescens and to the cellulose-binding protein p40 of Streptomyces halstedii. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of Vibrio harveyi. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of P. aeruginosa, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D).

Immunogenicity of in vitro folded outer membrane protein PorA of Neisseria meningitidis.

In vitro folded and the denatured form of PorA P1.6 from Neisseria meningitidis strain M990 were used for immunization studies in mice. Previously, the antigen was isolated from cytoplasmic inclusion bodies, folded and purified. Its immunogenicity without adjuvant appeared to be low. The addition of the adjuvant QuilA, but not of galE lipooligosaccharide, considerably enhanced the immunogenicity. Moreover, when immunized with folded PorA P1.6 plus QuilA, a clear switch towards the IgG2a subclass of antibodies and concomitantly, the appearance of serum bactericidal activity, which is believed to be important for protective immunity, was observed. Hence, a tool for preparing vaccines against serogroup B meningococci devoid of endotoxin is available.

Association of iron-regulated outer membrane proteins of Neisseria meningitidis with the RmpM (class 4) protein.

The RmpM (class 4) protein of Neisseria meningitidis has previously been shown to be associated with the outer membrane porins. In the present study, we demonstrate that this protein forms complexes with the lactoferrin receptor LbpA, the transferrin receptor TbpA and the siderophore receptor FrpB as well. This complexation apparently resulted in a stabilization of oligomeric forms of these iron-regulated proteins. In vitro experiments further revealed a reduced ability to acquire iron from human lactoferrin in the rmpM mutant. Furthermore, all TonB-dependent receptors investigated here appeared to exist as oligomers (probably dimers), suggesting that this is a general feature of this class of proteins.