Site-directed mutagenesis studies to probe the role of specific residues in the external loop (L3) of OmpF and OmpC porins in susceptibility of Serratia marcescens to antibiotics.(Report)

Abstract: Serratia marcescens is a nosocomial bacterium with natural resistance to a broad spectrum of antibiotics, making treatment challenging. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, controlled in part by OmpF and OmpC porin proteins. To investigate the direct role of these porins in the diffusion of antibiotics across the outer membrane, we have created an ompF-ompC porin-deficient strain of S. marcescens. A considerable similarity between the S. marcescens porins and those from other members of Enterobacteriaceae was detected by sequence alignment, with the exception of a change in a conserved region of the third external loop (L3) of the S. marcescens OmpC protein. Serratia marcescens OmpC has aspartic acid instead of glycine in position 112, methionine instead of aspartic acid in position 114, and glutamine in position 124, while in S. marcescens OmpF this is a glycine at position 124. To investigate the role of amino acid positions 112, 114, and 124 and how the observed changes within OmpC porin may play a part in pore permeability, 2 OmpC sites were altered in the Enterobacteriaceae consensus (D112G and M114D) through site-directed mutagenesis. Also, Q124G in OmpC, G124Q in OmpF, and double mutants of these amino acid residues were constructed. Antibiotic accumulation assays and minimal inhibitory concentrations of the strains harboring the mutated porins were performed, while liposome swelling experiments were performed on purified porins. Our results demonstrate that the amino acid at position 114 is not responsible for either antibiotic size or ionic selection, the amino acid at position 112 is responsible for size selection only, and position 124 is involved in both size and ionic selection.

Key words: Serratia marcescens, antibiotic resistance, porins, site-directed mutagenesis.

Resume: Serratia marcescens est une bacterie nosocomiale possedant une resistance naturelle a un large spectre d'antibiotiques, rendant son traitement difficile. La permeabilite reduite de la membrane externe, controlee par les porines OmpF et OmpC, est un facteur qui contribue a cette resistance naturelle aux antibiotiques. Afin d'examiner le role direct de ces porines dans la diffusion des antibiotiques a travers la membrane externe, nous avons cree une souche de S. marcescens deficiente en ompF-ompC. Une similarite considerable a ete trouvee entre les porines de S. marcescens et celles d'autres membres des Enterobacteriaceae par un alignement de sequences, a l'exception d'un changement au sein d'une region conservee de la troisieme boucle externe (L3) de la proteine OmpC de S. marcescens. OmpC de S. marcescens possede un acide aspartique plutot qu'une glycine en position 112, une methionine plutot qu'un acide aspartique en position 114 et une glutamine en position 124, alors que dans OmpF de S. marcescens, ce residu est une glycine. Afin de determiner le role des acides amines situes en position 112, 114 et 124, et determiner comment les changements observes au sein de la porine OmpC peuvent contribuer a la permeabilite du pore, deux sites chez OmpC ont ete modifies a l'interieur de la sequence consensus des Enterobacteriaceae (D112G et M114D) par mutagenese dirigee. De meme, Q124G de OmpC et G124Q de OmpF, ainsi que des doubles mutants ont ete construits. Des essais d'accumulation d'antibiotiques et les concentrations minimale inhibitrice des souches exprimant ces porines mutees ont ete effectues, alors que des experiences de gonflement de liposomes ont ete realisees avec les porines purifiees. Nos resultats demontrent que l'acide amine situe en position 114 n'est ni responsable de la selection selon la taille de l'antibiotique ni de la selection ionique, l'acide amine situe en position 112 est responsable de la selection selon la taille alors que l'acide amine situe en position 124 est implique dans les deux types de selection, selon la taille et ionique.

Mots-cles: Serratia marcescens, resistance aux antibiotiques, porines, mutagenese dirigee.

[Traduit par la Redaction]

Introduction

Serratia marcescens is a Gram-negative enteric bacterium, which has become an important opportunistic pathogen associated with a number of life-threatening diseases and nosocomial infections, such as urinary tract infections, respiratory tract infections, meningitis, septicaemia, and wound infections (Hejazi and Falkiner 1997). This organism possesses a high intrinsic resistance to a variety of antibiotics including aminoglycosides, [beta]-lactams, first- and second-generation cephalosporins, and quinolones (Fujimaki et al. 1989; Kumar and Worobec 2003) making treatment very difficult. The basis of this antibiotic resistance is multi-factorial, involving different mechanisms for each group of antibiotics. For example, resistance to [beta]-lactam antibiotics is achieved by (i) outer membrane impermeability mediated by porins (Gutmann et al. 1984), (ii) [beta]-lactamases found in the periplasm that degrade these drugs (Sanders and Sanders 1992), and (iii) altered inner membrane penicillin-binding proteins, which are the target for these antibiotics (Gunkel et al. 1991). Likewise, resistance to quinolones can be a result of mutations in DNA gyrase (the target enzyme for this group of antibiotics) (Fujimaki et al. 1989) and (or) over-expression of multidrug resistance efflux pumps (Ma et al. 1994).

The multifactorial nature of antibiotic resistance was demonstrated in a study using Enterobacter aerogenes, a related nosocomial pathogen. Investigators reported a simultaneous change in membrane permeability because of porin deficiency concomitant with the expression of an efflux mechanism, which renders this bacterium resistant to both [beta]-lactam and fluoroquinolone antibiotics (Mallea et al. 1998). One of our objectives was to study the role of outer membrane permeability in the [beta]-lactam resistance of S. marcescens.

For [beta]-lactam antibiotics to be effective in Gram-negative bacteria they must penetrate the outer membrane. The Gram-negative outer membrane is a semipermeable lipid bilayer that behaves like a molecular sieve to allow the passage of small hydrophilic molecules, such as nutrients, waste products, and [beta]-lactam antibiotics (Nikaido 1994), into and out of the cell. The degree of permeability of this membrane depends on the presence of poreforming porin proteins, which are membrane-spanning molecules that form water-filled channels (Nikaido 1994). Channel size and hydrophilicity are determined by an eyelet loop of the polypeptide chain that extends into the pore (Nikaido 1994). Nonspecific porins are typically involved in the passage of [beta]-lactam and other families of antibiotics (e.g., Escherichia coli OmpF/C (Nakae 1976; Mizuno et al. 1983) or OprF of Pseudomonas aeruginosa (Woodruff and Hancock 1989)).

Two major nonspecific porins, OmpF and OmpC, have been identified in S. marcescens (Hutsul and Worobec 1994, 1997). Although the role of OmpF and OmpC in the antibiotic resistance of E. coli (Mortimer and Piddock 1993) and other members of Enterobacteriaceae, such as E. aerogenes and Klebsiella pneumoniae, has been studied extensively (Gutmann et al. 1984), little is known about S. marcescens porins. Changes in porin copy number, size, selectivity, or function can alter the …