Deep mutational scanning of the Neisseria meningitidis major pilin reveals the importance of pilus tip-mediated adhesion.

Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto-aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto-aggregation, pili must reach a minimum length to allow pilus-pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip-located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP-dependent aggregation and adhesion.

Inhibitors of the Neisseria meningitidis PilF ATPase provoke type IV pilus disassembly.

Despite the availability of antibiotics and vaccines, Neisseria meningitidis remains a major cause of meningitis and sepsis in humans. Due to its extracellular lifestyle, bacterial adhesion to host cells constitutes an attractive therapeutic target. Here, we present a high-throughput microscopy-based approach that allowed the identification of compounds able to decrease type IV pilus-mediated interaction of bacteria with endothelial cells in the absence of bacterial or host cell toxicity. Compounds specifically inhibit the PilF ATPase enzymatic activity that powers type IV pilus extension but remain inefficient on the ATPase that promotes pilus retraction, thus leading to rapid pilus disappearance from the bacterial surface and loss of pili-mediated functions. Structure activity relationship of the most active compound identifies specific moieties required for the activity of this compound and highlights its specificity. This study therefore provides compounds targeting pilus biogenesis, thereby inhibiting bacterial adhesion, and paves the way for a novel therapeutic option for meningococcal infections.

Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host.

In Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution--ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness.

Origin in Acinetobacter gyllenbergii and dissemination of aminoglycoside-modifying enzyme AAC(6')-Ih.

OBJECTIVES: The aac(6')-Ih gene encoding aminoglycoside 6'-N-acetyltransferase type I subtype h [AAC(6')-Ih] is plasmid-borne in Acinetobacter baumannii where it confers high-level amikacin resistance, but its origin remains unknown. We searched for the gene in the genomes of a collection of 133 Acinetobacter spp. and studied its species specificity, expression and dissemination. METHODS: Gene copy number was determined by quantitative PCR, expression by quantitative RT-PCR, MIC by microdilution and transfer by plasmid mobilization. RESULTS: The aac(6')-Ih gene was present in the chromosome of the two Acinetobacter gyllenbergii of the collection and was detected in all seven A. gyllenbergii clinical isolates. They had indistinguishable flanking regions indicating that the gene was intrinsic to this species. A. baumannii PIS Aba23 promoters were provided by insertion of ISAba23, which disrupted the Pnative promoter in A. gyllenbergii. Both types of promoters were similarly potent in Escherichia coli and A. baumannii. Aminoglycoside MICs for A. baumannii harbouring pIP1858 were higher than for A. gyllenbergii due to gene dosage. The non-self-transferable plasmid could be mobilized to other A. baumannii cells by the broad host range plasmid RP4. CONCLUSIONS: We have found the origin of aac(6')-Ih in A. gyllenbergii, a species isolated, although rarely, in humans, and documented that dissemination of this gene is restricted to the Acinetobacter genus.

Identification of 50 class D ß-lactamases and 65 Acinetobacter-derived cephalosporinases in Acinetobacter spp.

Whole-genome sequencing of a collection of 103 Acinetobacter strains belonging to 22 validly named species and another 16 putative species allowed detection of genes for 50 new class D ß-lactamases and 65 new Acinetobacter-derived cephalosporinases (ADC). All oxacillinases (OXA) contained the three typical motifs of class D ß-lactamases, STFK, (F/Y)GN, and K(S/T)G. The phylogenetic tree drawn from the OXA sequences led to an increase in the number of OXA groups from 7 to 18. The topologies of the OXA and RpoB phylogenetic trees were similar, supporting the ancient acquisition of blaOXA genes by Acinetobacter species. The class D ß-lactamase genes appeared to be intrinsic to several species, such as Acinetobacter baumannii, Acinetobacter pittii, Acinetobacter calcoaceticus, and Acinetobacter lwoffii. Neither blaOXA-40/143- nor blaOXA-58-like genes were detected, and their origin remains therefore unknown. The phylogenetic tree analysis based on the alignment of the sequences deduced from blaADC revealed five main clusters, one containing ADC belonging to species closely related to A. baumannii and the others composed of cephalosporinases from the remaining species. No indication of blaOXA or blaADC transfer was observed between distantly related species, except for blaOXA-279, possibly transferred from Acinetobacter genomic species 6 to Acinetobacter parvus. Analysis of ß-lactam susceptibility of seven strains harboring new oxacillinases and cloning of the corresponding genes in Escherichia coli and in a susceptible A. baumannii strain indicated very weak hydrolysis of carbapenems. Overall, this study reveals a large pool of ß-lactamases in different Acinetobacter spp., potentially transferable to pathogenic strains of the genus.

Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms.

Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cyro-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wetting.

The shape of cellular membranes is highly regulated by a set of conserved mechanisms that can be manipulated by bacterial pathogens to infect cells. Remodeling of the plasma membrane of endothelial cells by the bacterium Neisseria meningitidis is thought to be essential during the blood phase of meningococcal infection, but the underlying mechanisms are unclear. Here we show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a physical mechanism that we term 'one-dimensional' membrane wetting. We provide a theoretical model that describes the physical basis of one-dimensional wetting and show that this mechanism occurs in model membranes interacting with nanofibers, and in human cells interacting with extracellular matrix meshworks. We propose one-dimensional wetting as a new general principle driving the interaction of cells with their environment at the nanoscale that is diverted by meningococci during infection.

Contribution of resistance-nodulation-cell division efflux systems to antibiotic resistance and biofilm formation in Acinetobacter baumannii.

UNLABELLED: Acinetobacter baumannii is a nosocomial pathogen of increasing importance due to its multiple resistance to antibiotics and ability to survive in the hospital environment linked to its capacity to form biofilms. To fully characterize the contribution of AdeABC, AdeFGH, and AdeIJK resistance-nodulation-cell division (RND)-type efflux systems to acquired and intrinsic resistance, we constructed, from an entirely sequenced susceptible A. baumannii strain, a set of isogenic mutants overexpressing each system following introduction of a point mutation in their cognate regulator or a deletion for the pump by allelic replacement. Pairwise comparison of every derivative with the parental strain indicated that AdeABC and AdeFGH are tightly regulated and contribute to acquisition of antibiotic resistance when overproduced. AdeABC had a broad substrate range, including ß-lactams, fluoroquinolones, tetracyclines-tigecycline, macrolides-lincosamides, and chloramphenicol, and conferred clinical resistance to aminoglycosides. Importantly, when combined with enzymatic resistance to carbapenems and aminoglycosides, this pump contributed in a synergistic fashion to the level of resistance of the host. In contrast, AdeIJK was expressed constitutively and was responsible for intrinsic resistance to the same major drug classes as AdeABC as well as antifolates and fusidic acid. Surprisingly, overproduction of AdeABC and AdeIJK altered bacterial membrane composition, resulting in decreased biofilm formation but not motility. Natural transformation and plasmid transfer were diminished in recipients overproducing AdeABC. It thus appears that alteration in the expression of efflux systems leads to multiple changes in the relationship between the host and its environment, in addition to antibiotic resistance. IMPORTANCE: Increased expression of chromosomal genes for RND-type efflux systems plays a major role in bacterial multidrug resistance. Acinetobacter baumannii has recently emerged as an important human pathogen responsible for epidemics of hospital-acquired infections. Besides its remarkable ability to horizontally acquire resistance determinants, it has a broad intrinsic resistance due to low membrane permeability, endogenous resistance genes, and antibiotic efflux. The study of isogenic mutants from a susceptible A. baumannii clinical isolate overproducing or deleted for each of the three major RND-type pumps demonstrated their major contribution to intrinsic resistance and to the synergism between overproduction of an efflux system and acquisition of a resistance gene. We have also shown that modulation of expression of the structural genes for the efflux systems results in numerous alterations in membrane-associated cellular functions, in particular, in a decrease in biofilm formation and resistance gene acquisition.

Origin in Acinetobacter guillouiae and dissemination of the aminoglycoside-modifying enzyme Aph(3')-VI.

The amikacin resistance gene aphA6 was first detected in the nosocomial pathogen Acinetobacter baumannii and subsequently in other genera. Analysis of 133 whole-genome sequences covering the taxonomic diversity of Acinetobacter spp. detected aphA6 in the chromosome of 2 isolates of A. guillouiae, which is an environmental species, 1 of 8 A. parvus isolates, and 5 of 34 A. baumannii isolates. The gene was also present in 29 out of 36 A. guillouiae isolates screened by PCR, indicating that it is ancestral to this species. The Pnative promoter for aphA6 in A. guillouiae and A. parvus was replaced in A. baumannii by PaphA6, which was generated by use of the insertion sequence ISAba125, which brought a -35 sequence. Study of promoter strength in Escherichia coli and A. baumannii indicated that PaphA6 was four times more potent than Pnative. There was a good correlation between aminoglycoside MICs and aphA6 transcription in A. guillouiae isolates that remained susceptible to amikacin. The marked topology differences of the phylogenetic trees of aphA6 and of the hosts strongly support its recent direct transfer within Acinetobacter spp. and also to evolutionarily remote bacterial genera. Concomitant expression of aphA6 must have occurred because, contrary to the donors, it can confer resistance to the new hosts. Mobilization and expression of aphA6 via composite transposons and the upstream IS-generating hybrid PaphA6, followed by conjugation, seems the most plausible mechanism. This is in agreement with the observation that, in the recipients, aphA6 is carried by conjugative plasmids and flanked by IS that are common in Acinetobacter spp. Our data indicate that resistance genes can also be found in susceptible environmental bacteria. Importance: We speculated that the aphA6 gene for an enzyme that confers resistance to amikacin, the most active aminoglycoside for the treatment of nosocomial infections due to Acinetobacter spp., originated in this genus before disseminating to phylogenetically distant genera pathogenic for humans. Using a combination of whole-genome sequencing of a collection of Acinetobacter spp. covering the breadth of the known taxonomic diversity of the genus, gene cloning, detailed promoter analysis, study of heterologous gene expression, and comparative analysis of the phylogenetic trees of aphA6 and of the bacterial hosts, we found that aphA6 originated in Acinetobacter guillouiae, an amikacin-susceptible environmental species. The gene conferred, upon mobilization, high-level resistance to the new hosts. This work stresses that nonpathogenic bacteria can act as reservoirs of resistance determinants, and it provides an example of the use of a genomic library to study the origin and dissemination of an antibiotic resistance gene to human pathogens.

Homologous prime boosting based on intranasal delivery of non-pathogenic invasive Escherichia coli expressing MPT64, decreases Mycobacterium tuberculosis dissemination.

Protein-subunit vaccines as boosting strategies against tuberculosis (TB) infection are currently in the pipeline of TB vaccine research. Their main limitation is represented by their poor immunogenicity, which makes it necessary to couple protein-subunits with adjuvant molecules. In this study, we employed replication-deficient invasive Escherichia coli strains to deliver Mycobacterium tuberculosis proteins to the cytoplasm of non-phagocytic eukaryotic cells using various priming and prime-boosting vaccination protocols. Our results demonstrate that intranasal administration of invasive E. coli expressing the M. tuberculosis protective antigen MPT64 to mice primed with a recombinant BCG strain over-expressing MPT64 on its surface, decrease bacterial burden in mice spleens. Our data suggest that replication-deficient invasive E. coli may represent a suitable platform for BCG/rBCG priming followed by homologous-boosting immunization strategies.

Bacterial delivery of large intact genomic-DNA-containing BACs into mammalian cells.

Efficient delivery of large intact vectors into mammalian cells remains problematical. Here we evaluate delivery by bacterial invasion of two large BACs of more than 150 kb in size into various cells. First, we determined the effect of several drugs on bacterial delivery of a small plasmid into different cell lines. Most drugs tested resulted in a marginal increase of the overall efficiency of delivery in only some cell lines, except the lysosomotropic drug chloroquine, which was found to increase the efficiency of delivery by 6-fold in B16F10 cells. Bacterial invasion was found to be significantly advantageous compared with lipofection in delivering large intact BACs into mouse cells, resulting in 100% of clones containing intact DNA. Furthermore, evaluation of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene from its genomic locus, which was present in one of the BACs, showed that single copy integrations of the HPRT-containing BAC had occurred in mouse B16F10 cells and that expression of HPRT from each human copy was 0.33 times as much as from each endogenous mouse copy. These data provide new evidence that bacterial delivery is a convenient and efficient method to transfer large intact therapeutic genes into mammalian cells.

Wild-type intracellular bacteria deliver DNA into mammalian cells.

Gene transfer in vitro from intracellular bacteria to mammalian phagocytic and non-phagocytic cells and in vivo in mice has been reported. The bacteria used as DNA delivery vectors were engineered to lyze upon entry in the cell due to impaired cell wall synthesis for Shigella flexneri and invasive Escherichia coli, or production of a phage lysin for Listeria mono- cytogenes. In vivo gene transfer was obtained with attenuated Salmonella typhimurium and resulted in stimulation of mucosal immunity. We report that wild-type intracellular human pathogens, such as L. monocytogenes EGD or LO28 and S. flexneri M90T, mediate efficient in vitro transfer of functional genes into epithelial and macrophage cell lines. A low- efficiency transfer was obtained from strain EGD to mouse peritoneal macrophages. DNA transfer with S. typhimurium was observed only from atten-uated aroA strain SL7207 into COS-1 cell line. As demonstrated by the study of listeriolysin-defective L. monocytogenes or of S. typhimurium SL7207 aroA engineered to secrete listeriolysin, escape of bacteria or of plasmid DNA from the intracytoplasmic vacuole is required for transfer of genetic information to occur.

Eukaryotic promoters can direct protein synthesis in Gram-negative bacteria.

Intracellular bacteria can act as DNA delivery vectors into mammalian cells. Transfer of genetic information can be monitored by screening for cellular expression of a reporter gene under the control of an eukaryotic promoter. However, intracellular bacteria can also efficiently deliver heterologous proteins in the cell cytosol. We have studied the activity of the eukaryotic PCMV promoter in Escherichia coli and Salmonella typhimurium using the lacZ and gfp genes as reporters and determined its strength relative to those of PRSV and PSV40 in E. coli. We found substantial heterologous activity of fragments carrying the PCMV and PRSV promoters, the strength of PRSV being greater than that of PCMV, but did not detect any PSV40 activity in E. coli. The green fluorescent protein (GFP) synthesized in E. coli was transferred to COS-1 cells where it was detectable and stable. Insertion of a transcription terminator or deletion of the bacterial ribosome binding site downstream from PCMV led to the silencing of the promoter in bacteria but not in mammalian cells. These observations should incite to exert caution when interpreting data on the DNA transfer from bacteria to mammallian cells when the genes of interest are under the control of the PCMV or PRSV promoter.