Anindya S. Ghosh

Physiological functions of D-alanine carboxypeptidases in Escherichia coli

Bacterial cell shape is, in part, mediated by the peptidoglycan (murein) sacculus. Penicillin-binding proteins (PBPs) catalyze the final stages of murein biogenesis and are the targets of beta-lactam antibiotics. Several low molecular mass PBPs including PBP4, PBP5, PBP6 and DacD seem to possess DD-carboxypeptidase (DD-CPase) activity, but these proteins are dispensable for survival in laboratory culture. The physiological functions of DD-CPases in vivo are unresolved and it is unclear why bacteria retain these seemingly non-essential and enzymatically redundant enzymes. However, PBP5 clearly contributes to maintenance of cell shape in some PBP mutant backgrounds. In this review, we focus on recent findings concerning the physiological functions of the DD-CPases in vivo, identify gaps in the current knowledge of these proteins and suggest some possible courses for future study that might help reconcile current models of bacterial cell morphology.

Common β-lactamases inhibit bacterial biofilm formation

β‐Lactamases, which evolved from bacterial penicillin‐binding proteins (PBPs) involved in peptidoglycan (PG) synthesis, confer resistance to β‐lactam antibiotics. While investigating the genetic basis of biofilm development by Pseudomonas aeruginosa, we noted that plasmid vectors encoding the common β‐lactamase marker TEM‐1 caused defects in twitching motility (mediated by type IV pili), adherence and biofilm formation without affecting growth rates. Similarly, strains of Escherichia coli carrying TEM‐1‐encoding vectors grew normally but showed reduced adherence and biofilm formation, showing this effect was not species‐specific. Introduction of otherwise identical plasmid vectors carrying tetracycline or gentamicin resistance markers had no effect on biofilm formation or twitching motility. The effect is restricted to class A and D enzymes, because expression of the class D Oxa‐3 β‐lactamase, but not class B or C β‐lactamases, impaired biofilm formation by E. coli and P. aeruginosa. Site‐directed mutagenesis of the catalytic Ser of TEM‐1, but not Oxa‐3, abolished the biofilm defect, while disruption of either TEM‐1 or Oxa‐3 expression restored wild‐type levels of biofilm formation. We hypothesized that the A and D classes of β‐lactamases, which are related to low molecular weight (LMW) PBPs, may sequester or alter the PG substrates of such enzymes and interfere with normal cell wall turnover. In support of this hypothesis, deletion of the E. coli LMW PBPs 4, 5 and 7 or combinations thereof, resulted in cumulative defects in biofilm formation, similar to those seen in β‐lactamase‐expressing transformants. Our results imply that horizontal acquisition of β‐lactamase resistance enzymes can have a phenotypic cost to bacteria by reducing their ability to form biofilms. β‐Lactamases likely affect PG remodelling, manifesting as perturbation of structures involved in bacterial adhesion that are required to initiate biofilm formation.

Helical Disposition of Proteins and Lipopolysaccharide in the Outer Membrane of Escherichia coli

In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope of Escherichia coli displays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover. To determine if material in the side walls was organized in any way, we labeled outer membrane proteins with succinimidyl ester-linked fluorescent dyes and then grew the stained cells in the absence of dye. Labeled proteins were not evenly dispersed in the envelope but instead appeared as helical ribbons that wrapped around the outside of the cell. By staining the O8 surface antigen of E. coli 2443 with a fluorescent derivative of concanavalin A, we observed a similar helical organization for the lipopolysaccharide (LPS) component of the outer membrane. Fluorescence recovery after photobleaching indicated that some of the outer membrane proteins remained freely diffusible in the side walls and could also diffuse into polar domains. On the other hand, the LPS O antigen was virtually immobile. Thus, the outer membrane of E. coli has a defined in vivo organization in which a subfraction of proteins and LPS are embedded in stable domains at the poles and along one or more helical ribbons that span the length of this gram-negative rod.

Branching sites and morphological abnormalities behave as ectopic poles in shape‐defective Escherichia coli

Certain mutants in Escherichia coli lacking multiple penicillin‐binding proteins (PBPs) produce misshapen cells containing kinks, bends and branches. These deformed regions exhibit two structural characteristics of normal cell poles: the peptidoglycan is inert to dilution by new synthesis or turnover, and a similarly stable patch of outer membrane caps the sites. To test the premise that these aberrant sites represent biochemically functional but misplaced cell poles, we assessed the intracellular distribution of proteins that localize specifically to bacterial poles. Green fluorescent protein (GFP) hybrids containing polar localization sequences from the Shigella flexneri IcsA protein or from the Vibrio cholerae EpsM protein formed foci at the poles of wild‐type E. coli and at the poles and morphological abnormalities in PBP mutants. In addition, secreted wild‐type IcsA localized to the outer membrane overlying these aberrant domains. We conclude that the morphologically deformed sites in these mutants represent fully functional poles or pole fragments. The results suggest that prokaryotic morphology is driven, at least in part, by the controlled placement of polar material, and that one or more of the low‐molecular‐weight PBPs participate in this process. Such mutants may help to unravel how particular proteins are targeted to bacterial poles, thereby creating important biochemical and functional asymmetries.

Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of Escherichia coli

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.

Deletion of penicillin-binding protein 5 (PBP5) sensitises Escherichia coli cells to β-lactam agents

Escherichia coli penicillin-binding protein 5 (PBP5), a dd-carboxypeptidase encoded by the dacA gene, plays a key role in the maintenance of cell shape. Although PBP5 shares one of the highest copy numbers among the PBPs, it is not essential for cell survival. To determine the effect of this redundant PBP on beta-lactam antibiotic susceptibility, PBP5 was deleted from O-antigen-negative E. coli K-12 (CS109) and O8-antigen-positive E. coli 2443, thus creating strains AM15-1 and AG1O5-1, respectively. Compared with the parent strains, both mutants were four- to eight-fold more susceptible to all the beta-lactam antibiotics tested. Reversion to beta-lactam resistance was observed in the mutants upon complementing with cloned PBP5, indicating the involvement of PBP5 in maintaining an O-antigen-independent intrinsic beta-lactam resistance in E. coli cells. To check whether other dacA homologues were able to substitute this behaviour of E. coli PBP5, AG1O5-1 was complemented with its nearest dacA homologues (Salmonella enterica serovar Typhimurium LT2, Vibrio cholerae and Haemophilus influenzae). All of the cloned homologues were capable of restoring the lost beta-lactam resistance in AG1O5-1, either completely or at least partially. Therefore, apart from maintaining cell shape, involvement of PBP5 in maintaining intrinsic beta-lactam resistance is an important physiological observation and we speculate that such a strategy of deleting PBP5 may be helpful to introduce beta-lactam susceptibility in the laboratory.

Sequences near the Active Site in Chimeric Penicillin Binding Proteins 5 and 6 Affect Uniform Morphology of Escherichia coli

Penicillin binding protein (PBP) 5, a DD-carboxypeptidase that removes the terminal D-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of Escherichia coli. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant, E. coli CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp(218) and Lys(219) residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of E. coli PBP 5.

PBP5, PBP6 and DacD play different roles in intrinsic β-lactam resistance of Escherichia coli.

Escherichia coli PBP5, PBP6 and DacD, encoded by dacA, dacC and dacD, respectively, share substantial amino acid identity and together constitute ~50 % of the total penicillin-binding proteins of E. coli. PBP5 helps maintain intrinsic β-lactam resistance within the cell. To test if PBP6 and DacD play simlar roles, we deleted dacC and dacD individually, and dacC in combination with dacA, from E. coli 2443 and compared β-lactam sensitivity of the mutants and the parent strain. β-Lactam resistance was complemented by wild-type, but not dd-carboxypeptidase-deficient PBP5, confirming that enzymic activity of PBP5 is essential for β-lactam resistance. Deletion of dacC and expression of PBP6 during exponential or stationary phase did not alter β-lactam resistance of a dacA mutant. Expression of DacD during mid-exponential phase partially restored β-lactam resistance of the dacA mutant. Therefore, PBP5 dd-carboxypeptidase activity is essential for intrinsic β-lactam resistance of E. coli and DacD can partially compensate for PBP5 in this capacity, whereas PBP6 cannot.

A weak DD-carboxypeptidase activity explains the inability of PBP 6 to substitute for PBP 5 in maintaining normal cell shape in Escherichia coli.

Penicillin-binding protein (PBP) 5 plays a critical role in maintaining normal cellular morphology in mutants of Escherichia coli lacking multiple PBPs. The most closely related homologue, PBP 6, is 65% identical to PBP 5, but is unable to substitute for PBP 5 in returning these mutants to their wild-type shape. The relevant differences between PBPs 5 and 6 are localized in a 20-amino acid stretch of domain I in these proteins, which includes the canonical KTG motif at the active site. We determined how these differences affected the enzymatic properties of PBPs 5 and 6 toward beta-lactam binding and the binding and hydrolysis of two peptide substrates. We also investigated the enzymatic properties of recombinant fusion proteins in which active site segments were swapped between PBPs 5 and 6. The results suggest that the in vivo physiological role of PBP 5 is distinguished from PBP 6 by the higher degree of DD-carboxypeptidase activity of the former.

Silk fibroin nanoparticles support in vitro sustained antibiotic release and osteogenesis on titanium surface

UNLABELLED Increasing amounts of metal-based implants are used for orthopedic or dental surgeries throughout the world. Still several implant-related problems such as inflammation, loosening and bacterial infection are prevalent. These problems stem from the immediate microbial contamination and failure of initial osteoblast adhesion. Additionally, bacterial infections can cause serious and life-threatening conditions such as osteomyelitis. Here, antibiotic (gentamicin)-loaded silk protein fibroin (non-mulberry silkworm, Antheraea mylitta) nanoparticles are fabricated and deposited over the titanium surface to achieve sustained drug release in vitro and to alter the surface nano-roughness. Based on the altered surface topography, chemistry and antibacterial activity, we conclude that the nanoparticle-deposited surfaces are superior for osteoblast adhesion, proliferation and differentiation in comparison to bare Ti. This method can be utilized as a cost-effective approach in implant modification. FROM THE CLINICAL EDITOR Titanium-based implants are commonly used in the field of orthopedics or dentistry. Surface modification of an implant is vital to ensure osseointegration. In this article, the author investigated the use of silk protein fibroins for metal surface modification and also for drug delivery against bacteria. The encouraging data should provide a new method in terms of nanotechnology in the respective clinical fields.