Phage resistance mutation triggered by OmpC deficiency in Klebsiella pneumoniae induced limited fitness costs.

Outer membrane proteins (OMPs) play an important role in bacterial fitness costs. Derived from the interaction between Klebsiella pneumoniae K7 and phage GH-K3, K7RB is an outer membrane porin-deficient phage-resistant mutant strain triggered by ompC712 deletion, exhibits expression inhibition of OmpC, OmpN, KPN_02430 and OmpF, but its fitness costs and regulatory mechanism remains unknown. In this study, compared with K7, K7RB showed almost unaffected growth rate, slightly decreased virulence, and increased resistance to some antibiotics. Transcriptome analysis showed that the pathways of glycerolipid metabolism and nitrogen metabolism in K7RB were significantly inhibited, while the transcription of permeases belonging to ABC transporters tended to be active, nutrient uptakes such as citrate and phenylalanine were also enhanced. However, transcriptional up-regulation in K7RB was inhibited by overexpression of OmpC, OmpN, KPN_02430 and OmpF in general. Overexpression of OmpN, KPN_02430 and OmpF, respectively, restoring the sensitivity of strains to antibiotics to varying degrees, while OmpC overexpression aggravated the bacterial drug-resistance especially to ß-lactam antibiotics. Besides, unlike OmpC and OmpF, overexpression of OmpN and KPN_02430 reduced bacterial virulence. In brief, by revealing the limited fitness costs of phage-resistant mutant K. pneumoniae with porin-deficiency, our study providing a reference for the design and development of drugs to inhibit the ways of bacterial metabolic rewiring and to increase fitness costs.

Simulation of pH-Dependent, Loop-Based Membrane Protein Gating Using Pretzel.

Bacterial porins often exhibit ion conductance and gating behavior which can be modulated by pH. However, the underlying control mechanism of gating is often complex, and direct inspection of the protein structure is generally insufficient for full mechanistic understanding. Here we describe Pretzel, a computational framework that can effectively model loop-based gating events in membrane proteins. Our method combines Monte Carlo conformational sampling, structure clustering, ensemble energy evaluation, and a topological gating criterion to model the equilibrium gating state under the pH environment of interest. We discuss details of applying Pretzel to the porin outer membrane protein G (OmpG).

Effect of helical kink in antimicrobial peptides on membrane pore formation.

Every cell is protected by a semipermeable membrane. Peptides with the right properties, for example Antimicrobial peptides (AMPs), can disrupt this protective barrier by formation of leaky pores. Unfortunately, matching peptide properties with their ability to selectively form pores in bacterial membranes remains elusive. In particular, the proline/glycine kink in helical peptides was reported to both increase and decrease antimicrobial activity. We used computer simulations and fluorescence experiments to show that a kink in helices affects the formation of membrane pores by stabilizing toroidal pores but disrupting barrel-stave pores. The position of the proline/glycine kink in the sequence further controls the specific structure of toroidal pore. Moreover, we demonstrate that two helical peptides can form a kink-like connection with similar behavior as one long helical peptide with a kink. The provided molecular-level insight can be utilized for design and modification of pore-forming antibacterial peptides or toxins.

Biology of the Gonococcus: Disease and Pathogenesis.

Neisseria gonorrhoeae infection is a major public health problem worldwide. The increasing incidence of gonorrhea coupled with global spread of multidrug-resistant isolates of gonococci has ushered in an era of potentially untreatable infection. Gonococcal disease elicits limited immunity, and individuals are susceptible to repeated infections. In this chapter, we describe gonococcal disease and epidemiology and the structure and function of major surface components involved in pathogenesis. We also discuss the mechanisms that gonococci use to evade host immune responses and the immune responses following immunization with selected bacterial components that may overcome evasion. Understanding the biology of the gonococcus may aid in preventing the spread of gonorrhea and also facilitate the development of gonococcal vaccines and treatments.

The fitness costs and trade-off shapes associated with the exclusion of nine antibiotics by OmpF porin channels.

The trade-off relationship between antibiotic exclusion and nutrient access across the Gram-negative outer membrane is determined by structural constraints in porin channels. The precise nutritional cost of exclusion is unknown for different antibiotics, as are the shapes of the nutrition-susceptibility trade-off. Using a library of 10 engineered isogenic Escherichia coli strains with structural modifications of OmpF porin expressed at a constant level, susceptibilities were measured for nine antibiotics and the nutritional fitness costs estimated by competitions in chemostats. Different antibiotics exhibited a remarkably varied range of geometries in the nutrition-susceptibility trade-off, including convex, concave and sigmoidal trade-off shapes. The trade-off patterns predict the possibility of adaptations in contributing to antibiotic resistance; exclusion of amoxicillin or trimethoprim in ompF mutants can occur with little loss of fitness whereas kanamycin and streptomycin exclusion has a high cost. Some individual OmpF changes even allow positive correlations (trade-ups), resulting in increased fitness and decreased susceptibility specifically to cephalexin or ciprofloxacin. The surprising plasticity of the nutrition-exclusion relationship means that there are no generalisable rules that apply to decreasing susceptibility for all antibiotics. The protein changes are exquisitely specific in determining nutritional fitness and adaptive outcomes in a structural constraint trade-off.

Fitness Cost of Rifampin Resistance in Neisseria meningitidis: In Vitro Study of Mechanisms Associated with rpoB H553Y Mutation.

Rifampin chemoprophylaxis against Neisseria meningitidis infections led to the onset of rifampin resistance in clinical isolates harboring point mutations in the rpoB gene, coding for the RNA polymerase ß chain. These resistant strains are rare in medical practice, suggesting their decreased fitness in the human host. In this study, we isolated rifampin-resistant rpoB mutants from hypervirulent serogroup C strain 93/4286 and analyzed their different properties, including the ability to grow/survive in different culture media and in differentiated THP-1 human monocytes and to compete with the wild-type strain in vitro. Our results demonstrate that different rpoB mutations (H553Y, H553R, and S549F) may have different effects, ranging from low- to high-cost effects, on bacterial fitness in vitro. Moreover, we found that the S549F mutation confers temperature sensitivity, possibly explaining why it is observed very rarely in clinical isolates. Comparative high-throughput RNA sequencing analysis of bacteria grown in chemically defined medium demonstrated that the low-cost H553Y substitution resulted in global transcriptional changes that functionally mimic the stringent response. Interestingly, many virulence-associated genes, including those coding for meningococcal type IV pili, porin A, adhesins/invasins, IgA protease, two-partner secretion system HrpA/HrpB, enzymes involved in resistance to oxidative injury, lipooligosaccharide sialylation, and capsular polysaccharide biosynthesis, were downregulated in the H553Y mutant compared to their level of expression in the wild-type strain. These data might account for the reduced capacity of this mutant to grow/survive in differentiated THP-1 cells and explain the rarity of H553Y mutants among clinical isolates.

Amelioration of the Fitness Costs of Antibiotic Resistance Due To Reduced Outer Membrane Permeability by Upregulation of Alternative Porins.

The fitness cost of antibiotic resistance is a key parameter in determining the evolutionary success of resistant bacteria. Studies of the effect of antibiotic resistance on bacterial fitness are heavily biased toward target alterations. Here we investigated how the costs in the form of a severely impaired growth rate associated with resistance due to absence of two major outer membrane porins can be genetically compensated. We performed an evolution experiment with 16 lineages of a double mutant of Escherichia coli with the ompCF genes deleted, and reduced fitness and increased resistance to different classes of antibiotics, including the carbapenems ertapenem and meropenem. After serial passage for only 250 generations, the relative growth rate increased from 0.85 to 0.99 (susceptible wild type set to 1.0). Compensation of the costs followed two different adaptive pathways where upregulation of expression of alternative porins bypassed the need for functional OmpCF porins. The first compensatory mechanism involved mutations in the phoR and pstS genes, causing constitutive high-level expression of the PhoE porin. The second mechanism involved mutations in the hfq and chiX genes that disrupted Hfq-dependent small RNA regulation, causing overexpression of the ChiP porin. Although susceptibility was restored in compensated mutants with PhoE overexpression, evolved mutants with high ChiP expression maintained the resistance phenotype. Our findings may explain why porin composition is often altered in resistant clinical isolates and provide new insights into how bypass mechanisms may allow genetic adaptation to a common multidrug resistance mechanism.

Genetic Lineages and Antimicrobial Resistance in Pseudomonas spp. Isolates Recovered from Food Samples.

Raw food is a reservoir of Pseudomonas isolates that could be disseminated to consumers. The presence of Pseudomonas spp. was studied in food samples, and the phenotypic and genotypic characterizations of the recovered isolates were analyzed. Two samples of meat (3%, turkey and beef) and 13 of vegetables (22%, 7 green peppers and 6 tomatoes) contained Pseudomonas spp. A total of 20 isolates were identified, and were classified as follows (number of isolates): P. aeruginosa (5), P. putida (5), P. nitroreducens (4), P. fulva (2), P. mosselli (1), P. mendocina (1), P. monteilii (1), and Pseudomonas sp. (1). These 20 Pseudomonas isolates were clonally different by pulsed-field-gel-electrophoresis, and were resistant to the following antibiotics: ticarcillin (85%), aztreonam (30%), cefepime (10%), imipenem (10%), and meropenem (5%), but were susceptible to ceftazidime, piperacillin, piperacillin-tazobactam, doripenem, gentamicin, tobramycin, amikacin, ciprofloxacin, norfloxacin, and colistin. Only one strain (Ps158) presented a class 1 integron lacking the 3' conserved segment. The five P. aeruginosa strains were typed by multilocus sequence typing in five different sequence-types (ST17, ST270, ST800, ST1455, and ST1456), and different mutations were detected in protein OprD that were classified in three groups. One strain (Ps159) showed a new insertion sequence (ISPa47) truncating the oprD gene, and conferring resistance to imipenem.

A kinetic Monte Carlo approach to investigate antibiotic translocation through bacterial porins.

Many relevant biological processes take place on time scales not reachable by standard all-atom computer simulations. The translocation of antibiotics through non-specific bacterial porins is an example. Microscopic effects compete to determine penetration routes and, consequently, free energy barriers to be overcome. Since bacteria can develop resistance to treatment also by reducing their antibiotic permeability, to understand the microscopic aspects of antibiotic translocation is an important step to rationalize drug design. Here, to investigate the translocation we propose a complete numerical model that combines the diffusion-controlled rate theory and a kinetic Monte Carlo scheme based on both experimental data and microscopically well-founded all-atom simulations. Within our model, an antibiotic translocating through an hour-glass-shaped channel can be described as a molecule moving on a potential of mean force featuring several affinity sites and a high central barrier. The implications of our results for the characterization of antibiotic translocation at in vivo concentrations are discussed. The presence of an affinity site close to the mouth of the channel seems to favor the translocation of antibiotics, the affinity site acting as a particle reservoir. Possible connections between results and the appearance of mutations in clinical strains are also outlined.

Mobility of BtuB and OmpF in the Escherichia coli outer membrane: implications for dynamic formation of a translocon complex.

Diffusion of two Escherichia coli outer membrane proteins-the cobalamin (vitamin B12) receptor (BtuB) and the OmpF porin, which are implicated in the cellular import pathways of colicins and phages-was measured in vivo. The lateral mobility of these proteins is relevant to the mechanism of formation of the translocon for cellular import of colicins such as the rRNase colicin E3. The diffusion coefficient (D) of BtuB, the primary colicin receptor, complexed to fluorescent antibody or colicin, is 0.05±0.01 µm2/s and 0.10±0.02 µm2/s, respectively, over a timescale of 25-150 ms. Mutagenesis of the BtuB TonB box, which eliminates or significantly weakens the interaction between BtuB and the TonB energy-transducing protein that is anchored in the cytoplasmic membrane, resulted in a fivefold larger value of D, 0.27±0.06 µm2/s for antibody-labeled BtuB, indicating a cytoskeletal-like interaction of TonB with BtuB. OmpF has a diffusion coefficient of 0.006±0.002 µm2/s, ∼10-fold smaller than that of BtuB, and is restricted within a domain of diameter 100 nm, showing it to be relatively immobile compared to BtuB. Thus, formation of the outer membrane translocon for cellular import of the nuclease colicins is a demonstrably dynamic process, because it depends on lateral diffusion of BtuB and collisional interaction with relatively immobile OmpF.

Deciphering ionic current signatures of DNA transport through a nanopore.

Within just a decade from the pioneering work demonstrating the utility of nanopores for molecular sensing, nanopores have emerged as versatile systems for single-molecule manipulation and analysis. In a typical setup, a gradient of the electrostatic potential captures charged solutes from the solution and forces them to move through a single nanopore, across an otherwise impermeable membrane. The ionic current blockades resulting from the presence of a solute in a nanopore can reveal the type of the solute, for example, the nucleotide makeup of a DNA strand. Despite great success, the microscopic mechanisms underlying the functionality of such stochastic sensors remain largely unknown, as it is not currently possible to characterize the microscopic conformations of single biomolecules directly in a nanopore and thereby unequivocally establish the causal relationship between the observables and the microscopic events. Such a relationship can be determined using molecular dynamics-a computational method that can accurately predict the time evolution of a molecular system starting from a given microscopic state. This article describes recent applications of this method to the process of DNA transport through biological and synthetic nanopores.

Fitness costs and stability of a high-level ciprofloxacin resistance phenotype in Salmonella enterica serotype enteritidis: reduced infectivity associated with decreased expression of Salmonella pathogenicity island 1 genes.

The fitness costs associated with high-level fluoroquinolone resistance were examined for phenotypically and genotypically characterized ciprofloxacin-resistant Salmonella enterica serotype Enteritidis mutants (104-cip and 5408-cip; MIC, >32 microg/ml). The stability of the fluoroquinolone resistance phenotype in both mutants was investigated to assess whether clones with better fitness could emerge in the absence of antibiotic selective pressure. Mutants 104-cip and 5408-cip displayed altered morphology on agar and by electron microscopy, reduced growth rates, motility and invasiveness in Caco-2 cells, and increased sensitivity to environmental stresses. Microarray data revealed decreased expression of virulence and motility genes in both mutants. Two clones, 104-revert and 1A-revertC2, with ciprofloxacin MICs of 3 and 2 microg/ml, respectively, were recovered from separate lineages of 104-cip after 20 and 70 passages, respectively, on antibiotic-free agar. All fitness costs, except motility, were reversed in 104-revert. Potential mechanisms associated with reversal of the resistance phenotype were examined. Compared to 104-cip, both 104-revert and 1A-revertC2 showed decreased expression of acrB and soxS but still overexpressed marA. Both acquired additional mutations in SoxR and ParC, and 1A-revertC2 acquired two mutations in MarA. The altered porin and lipopolysaccharide (LPS) profiles observed in 104-cip were reversed. In contrast, 5408-cip showed no reversal in fitness costs and maintained its high-level ciprofloxacin resistance for 200 passages on antibiotic-free agar. In conclusion, high-level ciprofloxacin resistance in S. Enteritidis is associated with fitness costs. In the absence of antibiotic selection pressure, isolates may acquire mutations enabling reversion to an intermediate-level ciprofloxacin resistance phenotype associated with less significant fitness costs.

Using multiconformation continuum electrostatics to compare chloride binding motifs in alpha-amylase, human serum albumin, and Omp32.

Ions are a ubiquitous component of the cellular environment, transferring into cells through membrane-embedded proteins. Ions bind to proteins to regulate their charge and function. Here, using multiconformation continuum electrostatics (MCCE), we show that the changes of chloride binding to alpha-amylase, human serum albumin (HSA) and Omp32 with pH, and of alpha-amylase with mutation agree well with experimental data. The three proteins represent three different types of binding. In alpha-amylase, chloride is bound in a specific buried site. Chloride binding is strongly coupled to the protonation state of a nearby lysine. MCCE calculates an 11-fold change in chloride affinity between the wild-type alpha-amylase and the K300R mutant, in good agreement with the measured 10-fold change.Without considering the coupled protonation reaction, the calculated affinity change would be more than 10(6)-fold. In HSA, chlorides are distributed on the protein surface. Although HSA has a negative net charge, it binds more anions than cations. There are no highly occupied binding sites in HSA. Rather, there are many partially occupied sites near clusters of basic residues. The relative affinity of bound ions of different charges is shown to depend on the distribution of charged residues on the surface rather than the overall net charge of the protein. The calculated strong pH dependence of the number of chlorides bound and the anion selectivity agree with those of previous experiments. In Omp32, chlorides are stabilized in an anion-selective transmembrane channel in a pH-independent manner. The positive electrostatic potential in Omp32 results in about two chlorides and no cations bound in the transmembrane region of this anion-selective channel. The studies here show that with the ability to sample multiple binding sites and coupled protein protonation states, MCCE provides a powerful tool to analyze and predict ion binding. The calculations overestimate the affinity of surface chloride in HSA and Omp32 relative to the buried ion in amylase. Differences between ion-solvent interactions for buried and surface ions will be discussed.

Steric selectivity in Na channels arising from protein polarization and mobile side chains.

Monte Carlo simulations of equilibrium selectivity of Na channels with a DEKA locus are performed over a range of radius R and protein dielectric coefficient epsilon(p). Selectivity arises from the balance of electrostatic forces and steric repulsion by excluded volume of ions and side chains of the channel protein in the highly concentrated and charged (approximately 30 M) selectivity filter resembling an ionic liquid. Ions and structural side chains are described as mobile charged hard spheres that assume positions of minimal free energy. Water is a dielectric continuum. Size selectivity (ratio of Na+ occupancy to K+ occupancy) and charge selectivity (Na+ to Ca2+) are computed in concentrations as low as 10(-5) M Ca2+. In general, small R reduces ion occupancy and favors Na+ over K+ because of steric repulsion. Small epsilon(p) increases occupancy and favors Na+ over Ca2+ because protein polarization amplifies the pore's net charge. Size selectivity depends on R and is independent of epsilon(p); charge selectivity depends on both R and epsilon(p). Thus, small R and epsilon(p) make an efficient Na channel that excludes K+ and Ca2+ while maximizing Na+ occupancy. Selectivity properties depend on interactions that cannot be described by qualitative or verbal models or by quantitative models with a fixed free energy landscape.

Intragenic suppressors of an OmpF assembly mutant and assessment of the roles of various OmpF residues in assembly through informational suppressors.

We employed two separate genetic approaches to examine the roles of various OmpF residues in assembly. In one approach, intragenic suppressors of a temperature-sensitive OmpF assembly mutant carrying a W214E substitution were sought at 42 degrees C, or at 37 degrees C in a genetic background lacking the periplasmic folding factor SurA. In the majority of cases (58 out of 61 revertants), the suppressors mapped either at the original site (position 214) or two residues downstream from it. In the remaining three revertants that were obtained in a surA background, an alteration of N230Y was located 16 residues away from the original site. The N230Y suppressor also corrected OmpF315 assembly at 42 degrees C in a surA(+) background, indicating that the two different physiological environments imposed similar assembly constraints. The specificity of N230Y was tested against five different residues at position 214 of mature OmpF. Clear specificity was displayed, with maximum suppression observed for the original substitution at position 214 (E214) against which the N230Y suppressor was isolated, and no negative effect on OmpF assembly was noted when the wild-type W214 residue was present. The mechanism of suppression may involve compensation for a specific conformational defect. The second approach involved the application of informational suppressors (Su-tRNA) in combination with ompF amber mutations to generate variant OmpF proteins. In this approach we targeted the Y40, Q66, W214, and Y231 residues of mature OmpF and replaced them with S, Q, L, and Y through the action of Su-tRNAs. Thus, a total of 16 variant OmpF proteins were generated, of which three were identical to the parental protein, and two variants carrying W214Q and Y231Q substitutions were similar to assembly-defective proteins isolated previously (R. Misra, J. Bacteriol. 175:5049-5056, 1993). The results obtained from these analyses provided useful information regarding the compatibility of various alterations in OmpF assembly.