Mutational Variation Analysis of oprD Porin Gene in Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa

Abstract

The present study deals with the outer membrane OprD porin protein in 29 clinical bacterial isolates of multidrug-resistant Pseudomonas aeruginosa. oprD porin gene expression was investigated using real-time reverse transcription-PCR. Amplicons from oprD and its transcriptional regulator mexT gene were sequenced and analyzed for mutations. Hypothetical models of selected mutant OprD-porin proteins were predicted and refined by homology modeling approach. oprD ampliconic sequences were also screened for restriction fragment length polymorphism (RFLP). The oprD gene was found to be downregulated in 89.7% (n = 26) of the isolates in comparison to the transcript levels in the reference strain P. aeruginosa—PAO (MTCC-3541). Interestingly, all these isolates displayed the presence of a conspicuous 8-bp deletion (GGCCAGCC) at nucleotide position 235 of mexT regulatory gene. Based on the mutational patterns observed in oprD gene, the isolates were classified into categories designated as A, B1-2, C1-4, D1-6, E1-2, and F. Our hypothetical models revealed that mutations were predominantly confined to the extracellular loops emanating from the β-barrel porin protein. These protein models also enabled clear visualization of loss of substantial portions of the truncated polypeptide. Incidentally, since most of the oprD amplicons of the clinical isolates were found to display distinct RFLP banding patterns, our results also provide a useful diagnostic tool for detection of P. aeruginosa porin mutants.

Introduction

P seudomonas aeruginosa —an opportunistic pathogen—can thrive well in many environments due to high versatility and adaptability. It has now achieved a superbug status by acquiring multidrug-resistant (MDR) phenotypes through intrinsic and acquired resistance mechanisms leading to treatment failures.^1,2 These infections are usually treated with aminoglycosides, β-lactams, and polymyxins.³ The alarming increase in the emergence and spread of P. aeruginosa strains resistant to first-line broad-spectrum antibiotics, including the carbapenem nonsusceptible ones, pose a serious health threat.^4,5 Carbapenem-uptake is known to occur mainly through OprD porin proteins. Recent studies indicate involvement of OprD downregulation along with overexpression of efflux systems in the development of such resistance.^6,7

Porins are outer membrane proteins which form water-filled pores with various grades of selectivity. They consist of transmembrane antiparallel β-strands with alternating hydrophobic and hydrophilic amino acids facing outward and inward, respectively, connected by short periplasmic turns and longer surface-exposed extracellular loops. They also act as molecular filters for hydrophilic substances and mediate transport of nutrients and ions across the membrane into the periplasm.^8,9

P. aeruginosa porin-OprD is a 45–49 kDa substrate-specific membrane protein which not only facilitates diffusion of basic amino acids, small peptides, and carbapenems into the cell but also functions as a serine protease.¹⁰ DNA and amino acid sequence identities of OprD among individual strains of P. aeruginosa range from 91% to 93% and 88% to 93%, respectively. Despite genetic variability among different strains, OprD shares close homology to the nonspecific Escherichia coli porin OmpF.^11,12 The X-ray crystal structure of OprD (PDB ID-2ODJ) was determined by Biswas et al.¹³

Expression of OprD is linked to both carbon and nitrogen metabolism of P. aeruginosa and is induced by arginine (mediated through regulatory protein ArgR), histidine, glutamate, or alanine.¹⁴ Mechanisms underlying OprD-dependent resistance involve mutations leading to decreased transcription of oprD or its translational disruption into functional protein.

Interference in oprD transcription can occur due to (1) disruption of its promoter as a result of insertion/deletions of IS elements (insertion sequences) (2) premature termination of transcription, (3) negative regulation by MexT, a regulatory protein of MexEF-OprN efflux pump, (4) salicylate-mediated repression, (5) negative regulation mediated through the regulatory proteins CzcR and CopR in the presence of zinc and copper, respectively, and (6) repression by two-component, ParR-ParS regulatory system known to be critical for multidrug resistance. Translational mechanisms that lead to OprD deficiency include (1) occurrence of frameshifts and premature stop codons due to mutations, insertions, and/or deletions, and (2) disruption of the structural gene by insertion of large IS elements.¹²

The present study aimed to analyze mutational variations in oprD porin gene and the consequent structural changes of the protein in MDR clinical isolates of P. aeruginosa collected from Kerala, the southernmost Indian state. Real-time reverse transcription-PCR technique was used to quantitate oprD gene transcription. Amplicons from oprD and its regulatory gene, mexT, were sequenced to analyze the mutational variants. The three-dimensional (3D) structure of the porin protein deducted from oprD amplicons were predicted by homology modeling approach. Hypothetical models of selected mutant OprD-porin proteins were constructed to depict the translational effects of the observed mutations. oprD amplicons were also analyzed for the presence of any restriction fragment length polymorphism (RFLP) arising due to gain or loss of restriction sites consequent to mutational differences among bacterial isolates.

Materials and Methods

Bacterial isolates and antimicrobial susceptibility testing

The present study involved a total of 29 clinical isolates of MDR P. aeruginosa, designated as Pa1–Pa29, collected during 2012–2016 from various clinical laboratories in Kerala, India. A reference strain, P. aeruginosa—PAO, Microbial Type Culture Collection—MTCC-3541, obtained from the Institute of Microbial Technology (IMTECH) Chandigarh, C.S.I.R. Govt. of India, was also used. Antibiotic sensitivity test was carried out by standard disc diffusion (Kirby-Bauer) assay on Mueller–Hinton agar plates based on the recommended guidelines¹⁵ using 17 antimicrobial discs (HiMedia, Mumbai, India): amikacin-30 μg (AK), ampicillin-10 μg (AMP), aztreonam-30 μg (AT), cefotaxime-30 μg (CTX), ceftazidime-30 μg (CAZ), cefepime-30 μg (CPM), chloramphenicol-30 μg (C), ciprofloxacin-5 μg (CIP), colistin-10 μg (CL), gentamicin-10 μg (GEN), imipenem- 10 μg (IPM), meropenem-10 μg (MRP), nalidixic acid-30 μg (NA), ofloxacin-5 μg (OF), piperacillin/tazobactam-100/10 μg (PIT), polymyxin-B-300 U (PB), and tetracycline-30 μg (TE).

Quantitative real-time PCR

Bacterial cultures were grown in Luria Bertani medium up to an O.D. of 2.0 at 600 nm, and total RNA was isolated using TRI Reagent^® (Sigma Aldrich) and their purity ascertained ensuring 260/280 O.D. ratios of ∼2.00. Traces of any residual DNA were removed by adding DNase I (Promega) according to the manufacturer's instructions. Synthesis of cDNA was performed as described by us earlier¹⁶ and stored at −20°C until use. Using cDNA templates, RT-PCR was performed using primers, purchased from Eurofins Genomics India Pvt. Ltd., (Bangalore, India) (Supplementary Table S1), for genes such as rpsL and oprD described previously.^17,18 Quantifications of cDNAs were carried out using SYBR Green PCR Master Mix (TaKaRa, Inc., Japan) in an Illumina Eco™ Real-Time PCR system. Relative expression of oprD gene was evaluated by the CT method¹⁹ using the constitutively expressed rpsL housekeeping gene transcript. Reference strain P. aeruginosa, PAO (MTCC-3541), was used for normalization of relative mRNA levels.

Amplification of genomic DNA for sequencing of mexT and oprD genes

Genomic DNA from individual bacterial cultures was isolated essentially as described by Ausubel et al.²⁰ The PCRs were performed with 100 ng of genomic DNA as template, using 2× Emerald GT Master Mix (TaKaRa, Inc.) in a minicycler (MJ Research). Primers used for DNA amplifications and sequencing (Eurofins Genomics India Pvt. Ltd.) are listed in Supplementary Table S1.^21,22 PCR products were cloned in E. coli DH5α using CloneJET PCR Cloning Kit according to the manufacturer's instructions. The recombinant plasmids were isolated from the transformants. These plasmids were sent to commercial facility (SciGenom Labs Pvt. Ltd., Kochi, India) to obtain the sequence information. The mexT and oprD ampliconic sequences were analyzed by NCBI BLAST by comparison with sequences of the reference strain, P. aeruginosa PAO1 from the GenBank database (Accession no. AE004091.2). Deduction of the amino acid sequences of variant OprD proteins were performed using ORF Finder and the sequences were aligned and analyzed using Clustal Omega.

Molecular modeling of OprD protein

The 3D structures of the channel protein OprD porin from the reference strain, P. aeruginosa PAO1 and the four mutant isolates, Pa12, Pa7, Pa25, and Pa27, were predicted by homology modeling. The target amino acid sequence of OprD protein (reference strain) was submitted to BLAST and searched against PDB database to identify the best homologous templates based on the percentage of identity, E-value, query coverage, total score, and similarity percentage. The atomic coordinates, alignment, and script files were prepared and initial model was predicted by Modeller 9.18, as per standard protocol.²³ The hypothetical model was energy minimized by ModRefiner tool.²⁴ The structure annotated pairwise alignment, and superimposition of the target and templates were performed by Superpose server²⁵ and the root mean square deviation (RMSD) was estimated to check the structural deviation in the backbone between the target and template.

The stereochemical quality of the structure was evaluated using Ramachandran plot generated by PROCHECK.²⁶ The hypothetical model was further refined by various computational biology tools for force fields such as ANOLEA,²⁷ QMEAN,²⁸ GROMOS,²⁹ and VERIFY 3D, and the secondary structure was predicted by DSSP.³⁰ The final hypothetical model of OprD porin of P. aeruginosa PAO1 was visualized by PyMOL.³¹ The structures of the four selected mutants, Pa12, Pa7, Pa25, and Pa27 representing different OprD types were modeled and refined by similar computational biology tools.

Restriction digestion

Restriction digestion reactions were set up in a total reaction volume of 10.0 μL containing 5.0 μL of the initial 25.0 μL oprD gene amplification reaction. Three such reaction sets were separately restricted (1.0 U of enzyme/μg DNA) with each of HincII, PstI, and PvuII restriction enzymes in the corresponding 1 × restriction buffers (Bangalore Genei, Pvt. Ltd.). The reaction mixtures were then incubated at the prescribed temperature (37°C) for 1 hr. The restriction digests were electrophoresed on 1% agarose gels followed by visualization and photography on a gel documentation system. For detection of the putative restriction sites within the PCR amplicons obtained from the oprD gene, NEB cutter V2.0³² was utilized.

Nucleotide sequence accession numbers

Nucleotide sequences obtained in the present study were deposited in the GenBank database under the following accession numbers: mexT gene sequences MH397275-MH397298; oprD gene sequences MH122946-MH122954, MH135303-MH135311, and MH142581-MH142588.

Results

Antimicrobial susceptibility

The antibiotic resistance profiles of clinical bacterial isolates are given in Table 1. The multiple antibiotic resistance index value of the clinical isolates, tested against 17 antibiotics and computed as described earlier,³³ were found to range from 0.5 to 1.0.

Table 1.

Antibiotic Resistance Profile of Pseudomonas aeruginosa Isolates from Different Specimen Types

Isolates	Specimen type^*	Antibiotic resistance profile
Isolates	Specimen type^*	AMP	CTX	CAZ	CPM	AT	IPM	MRP	AK	GEN	TE	C	NA	CIP	OF	PB	CL	PIT
Pa1^a	Urine	R	R	R	R	R	S	R	R	R	I	R	R	I	R	S	S	R
Pa2^a	Other body fluid	R	R	R	I	I	S	R	R	R	R	R	R	R	R	S	R	I
Pa3^a	Urine	R	R	R	R	R	R	I	R	R	R	R	R	R	R	S	S	R
Pa4^b	Other body fluid	R	R	R	S	I	R	S	S	R	R	R	R	I	S	S	R	I
Pa5^b	Other body fluid	R	R	R	R	I	S	S	S	I	R	R	R	I	I	S	R	R
Pa6^b	Other body fluid	R	R	R	S	I	I	S	I	R	R	R	R	S	I	S	R	S
Pa7^b	Other body fluid	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R
Pa8^b	Urine	R	R	R	R	I	R	R	R	R	R	I	R	R	R	S	R	R
Pa9^b	Other body fluid	R	R	S	R	I	S	R	R	R	R	R	R	R	R	R	R	R
Pa10^b	Urine	R	R	R	R	R	R	R	R	R	R	S	R	R	R	S	R	R
Pa11^b	Urine	R	R	I	R	R	R	R	R	R	R	R	R	R	R	R	R	R
Pa12^b	Sputum	R	R	R	R	R	S	R	R	R	R	R	R	R	R	R	R	I
Pa13^b	Other body fluid	R	R	R	R	I	R	R	R	R	R	R	R	R	R	S	R	I
Pa14^c	Urine	R	R	R	I	I	R	I	S	S	R	R	R	I	I	S	R	R
Pa15^c	Urine	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R
Pa16^b	Other body fluid	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R
Pa17^b	Other body fluid	R	R	I	R	R	S	R	R	R	R	R	R	R	R	S	R	R
Pa18^b	Urine	R	R	R	R	R	R	R	R	I	R	R	R	R	R	R	R	R
Pa19^b	Urine	R	R	R	R	R	R	R	R	R	R	S	R	R	R	R	R	R
Pa20^b	Other body fluid	R	R	R	R	R	S	R	R	R	R	R	R	R	R	R	R	R
Pa21^b	Other body fluid	R	I	R	R	S	R	R	S	R	R	R	R	R	R	R	R	I
Pa22^b	Other body fluid	R	R	R	R	R	S	R	R	R	R	R	R	R	R	S	R	R
Pa23^b	Other body fluid	R	R	R	R	R	S	R	R	R	R	R	R	R	R	R	R	R
Pa24^b	Other body fluid	R	R	R	R	R	S	R	R	R	R	R	R	R	R	R	R	R
Pa25^b	Urine	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R
Pa26^b	Urine	R	R	R	R	R	S	R	R	R	R	R	R	R	R	R	R	R
Pa27^b	Sputum	R	R	S	R	I	R	R	S	R	R	R	R	S	R	S	R	R
Pa28^a	Urine	R	R	R	R	R	S	R	R	R	R	R	R	R	R	S	R	R
Pa29^a	Urine	R	R	R	R	R	R	R	R	R	R	R	R	R	R	S	R	R

^* Names and addresses of clinical laboratories are not mentioned based on their request; patient details are also not being revealed due to ethical reasons. Three categories of specimen types, isolated from “urine,” “sputum,” and “other body fluids” were received by us as pure cultures.

Superscripts “a” and “b” denote isolates collected from clinical laboratory-1 and 2 (Calicut district, Kerala, India) during the period 2012–2016 and 2012–2014 respectively, while “c” denotes those collected from a clinical laboratory situated in Ernakulam district (Kerala, India) during 2012–2013.

Resistance profile of carbapenem antibiotics (IPM and MRP)–major substrate of OprD porin protein–are shown in boldface; R, I, and S denote resistant, intermediate, and sensitive phenotypes, respectively.

The antibiotic discs used for profiling: AMP, ampicillin; CTX, cefotaxime; CAZ, ceftazidime; CPM, cefepime AT-aztreonam; IPM, imipenem; MRP, meropenem; AK, amikacin; GEN, gentamicin; TE, tetracycline; C, chloramphenicol; NA, nalidixic acid; CIP, ciprofloxacin; OF, ofloxacin; PB, polymyxin-B; CL, colistin; PIT, piperacillin/tazobactam.

Porin—oprD gene expression analysis

Carbapenems are the last resort drugs used for treatment of MDR P. aeruginosa infections. One of the mechanisms underlying resistance developed against this β-lactam class of antibiotics involves downregulation of porin-OprD. Results obtained in the present study reveal that oprD remained downregulated in as many as 89.7% (n = 26) of the isolates (Table 2) barring three strains, Pa1, Pa11, and Pa19, with transcriptions nearly comparable to that of the reference strain, PAO. It is relevant here to note the observation of Xavier et al.³⁴ that reduced transcriptional levels of the oprD gene can be considered to be significant if found ≤70% compared to that of their reference strain.

Table 2.

Relative Decrease in oprD Gene Transcriptional Levels of P. aeruginosa Isolates

Isolates	Relative expression^* of oprD	Isolates	Relative expression^* of oprD
Pa1	0.77	Pa16	ND
Pa2	0.12	Pa17	0.03
Pa3	0.08	Pa18	ND
Pa4	0.07	Pa19	1.05
Pa5	0.10	Pa20	0.21
Pa6	ND	Pa21	0.09
Pa7	0.05	Pa22	ND
Pa8	ND	Pa23	0.13
Pa9	0.02	Pa24	ND
Pa10	0.29	Pa25	ND
Pa11	1.43	Pa26	0.48
Pa12	0.16	Pa27	0.08
Pa13	ND	Pa28	0.09
Pa14	ND	Pa29	0.06
Pa15	0.05	Pa29	0.06

^* Expression levels of porin oprD in P. aeruginosa isolates relative to that observed in the reference strain PAO assigned with a value of 1.0. Decrease in expression levels ≤0.7-fold compared to reference are indicated in boldface.

ND—amplicons not detected for reasons which remain unclear; this could probably be due to either extremely low levels of amplification or failure of expression.

Mutational variations of mexT regulatory gene

The mexT gene-specific amplicons were obtained with genomic-PCR of 24 out of 26 P. aeruginosa isolates with downregulated OprD except in Pa2 and Pa6 as reported by us earlier.¹⁶ Primers used for amplification were confined to the N-terminal portion encoded by the 5′ end of the mexT gene, which included the 8 bp insert involved in inactivation of the transcriptional activator. Interestingly, a comparative analysis of DNA sequences of the amplicons with that of P. aeruginosa-PAO1 (GenBank accession no. AE004091.2) revealed the occurrence of an 8-bp deletion (GGCCAGCC) at nucleotide position 235 within a 14-bp direct repeat in all isolates shown in Table 3. Furthermore, the results of BLASTN analyses of all mexT ampliconic sequences, which were found to be identical with the P. aeruginosa sequence deposits in GenBank, are included in Supplementary Table S2.

Table 3.

Mutations Observed in mexT Amplicons from P. aeruginosa Isolates

Mutations	Isolates
Isolates bearing 8 bp deletion^*	Pa3, Pa4, Pa5, Pa8, Pa9, Pa13, Pa17, Pa20, Pa21, Pa22, Pa24, and Pa28
Isolates bearing additional mutations upstream of the 8 bp deletion^*
⁶⁰Proline→Serine	Pa7, Pa10, Pa12, Pa14, Pa15, Pa16, Pa23, Pa25, Pa26, Pa27, and Pa29
⁶⁶Arginine→Histidine	Pa18

8 bp (²³⁵GGCCAGCC) deletion leading to frameshift mutations.

Mutational variations of oprD gene

To detect mutations in the oprD gene encoding porin channel protein, the amplicons derived from the 26 isolates exhibiting porin downregulation were sequenced. DNA sequences and the corresponding amino acids encoded were then compared with the sequences of the reference strain PAO1. On the basis of mutational patterns of the oprD gene, the isolates were classified into several types such as A, B1-2, C1-4, D1-6, E1-2, and F (Fig. 1 and Table 4). oprD type-A was categorized as wild type, in which both the nucleotide and amino acid sequences were found to be identical with the reference strain.

FIG. 1.

Amino acid sequences of Pseudomonas aeruginosa OprD types aligned with that of the reference strain—P. aeruginosa PAO1. Amino acid changes/deletions are highlighted in gray. The loop regions as determined by the crystal structure of the porin protein are demarcated with a thick gray line over the amino acid sequences of P. aeruginosa PAO1.⁵³

Table 4.

Different OprD Types Detected Among P. aeruginosa Isolates

OprD types	Isolates	Classification of OprD types	Alterations/mutations^* and similarity with GenBank deposits
A	Pa2, Pa6, Pa15, Pa18	Wild type	None
B1	Pa9	Full-length type with single amino acid substitution	V₁₂₇L
B1	Pa9		Similar to GenBank accession no. ETD93478.1.³⁵
B2	Pa29		G₃₁₆D
B2	Pa29		Similar to GenBank accession no. WP_101516673.1.
C1	Pa4	Full-length type with several polymorphisms	T₁₀₃S,K₁₁₅T,F₁₇₀L
C2	Pa12, Pa20, Pa22		T₁₀₃S,K₁₁₅T,F₁₇₀L,E₁₈₅Q, P₁₈₆G,V₁₈₉T, R₃₁₀E, A₃₁₅G,G₄₂₅A
C3	Pa13		T₁₀₃S,K₁₁₅T,F₁₇₀L,E₁₈₅Q, P₁₈₆G,V₁₈₉T,G₂₉₉S, R₃₁₀E,A₃₁₅G, E₃₉₆G,G₄₂₅A
C4	Pa21		W₆₅R,T₁₀₃S,K₁₁₅T,F₁₇₀L, E₁₈₅Q,P₁₈₆G,V₁₈₉T, K₂₀₅Q,R₃₁₀E,A₃₁₅G,G₄₂₅A
D1	Pa16	Several polymorphisms along with deletions and substitutions	F₂₆L,V₁₂₇L,E₁₈₅Q,P₁₈₆G, V₁₈₉T,E₂₀₂Q,I₂₁₀A, E₂₃₀K,S₂₄₀T,N₂₆₂T,T₂₇₆A, A₂₈₁G,K₂₉₆Q,Q₃₀₁E, R₃₁₀E, G₃₁₂R, A₃₁₅G,L₃₄₇M, S₄₀₃A,Q₄₂₄E
D1	Pa16		₃₇₂(VDSSSS -YAGL-)₃₈₃
D2	Pa26		S₅₇E,S₅₉R,W₆₅R,V₁₂₇L, E₁₈₅Q,P₁₈₆G,V₁₈₉T,E₂₀₂Q, I₂₁₀A,E₂₃₀K,S₂₄₀T,N₂₆₂T, A₂₈₁G,K₂₉₆Q,Q₃₀₁E, R₃₁₀E,A₃₁₅G,L₃₄₇M, S₄₀₃A,W₄₁₇R,Q₄₂₄E
D2	Pa26		₃₇₂(VDSSSS-YAGL-)₃₈₃
D3	Pa7, Pa14, Pa17, Pa24, Pa28		S₅₇E,S₅₉R,V₁₂₇L,E₁₈₅Q, P₁₈₆G,V₁₈₉T,E₂₀₂Q,I₂₁₀A, E₂₃₀K,S₂₄₀T,N₂₆₂T,T₂₇₆A, A₂₈₁G,K₂₉₆Q,Q₃₀₁E, R₃₁₀E,A₃₁₅G,L₃₄₇M, S₄₀₃A,Q₄₂₄E
			₃₇₂(VDSSSS-YAGL-)₃₈₃
			Similar to GenBank accession nos. ETD51009.1.³⁵ KAJ07311.1,³⁶ and ESQ66005.1³⁷
D4	Pa8		V₁₂₇L,E₁₈₅Q,P₁₈₆G,V₁₈₉T, E₂₀₂Q,I₂₁₀A,E₂₃₀K, S₂₄₀T, N₂₆₂T,T₂₇₆A,A₂₈₁G,K₂₉₆Q, Q₃₀₁E,R₃₁₀E, G₃₁₂R, A₃₁₅G, W₃₃₉R, L₃₄₇M, S₄₀₃A,Q₄₂₄E
D4	Pa8		₃₇₂(VDSSSS-YAGL-)₃₈₃
D5	Pa5, Pa23		V₁₂₇L,E₁₈₅Q,P₁₈₆G,V₁₈₉T, E₂₀₂Q,I₂₁₀A,E₂₃₀K,S₂₄₀T, N₂₆₂T,T₂₇₆A,A₂₈₁G,K₂₉₆Q, Q₃₀₁E, R₃₁₀E, G₃₁₂R, A₃₁₅G, L₃₄₇M, S₄₀₃A,Q₄₂₄E
			₃₇₂(VDSSSS-YAGL-)₃₈₃
			Similar to GenBank accession nos. KAJ11220.1,³⁶ KSC 28238.1³⁸ and AAS18314.2.³⁹
D6	Pa3		V₁₂₇L,E₁₈₅Q,P₁₈₆G,V₁₈₉T,E₂₀₂Q,I₂₁₀A,E₂₃₀K,S₂₄₀T,N₂₆₂T, T₂₇₆A,A₂₈₁G,K₂₉₆Q,Q₃₀₁E, G₃₁₂R,A₃₁₅G,L₃₄₇M,S₄₀₃A, Q₄₂₄E
D6	Pa3		₃₇₂(VDSSSS-YAGL-)₃₈₃
E1	Pa25	Several polymorphisms and premature stop codon	D₄₃N,S₅₇E,S₅₉R,E₂₀₂Q,I₂₁₀A,E₂₃₀K
E1	Pa25		TAT₇₀₈→TAG (stop codon)
E2	Pa10		V₁₂₇L,E₁₈₅Q,P₁₈₆G,V₁₈₉T, E₂₀₂Q,I₂₁₀A,E₂₃₀K,S₂₄₀T, N₂₆₂T,T₂₇₆A,A₂₈₁G,K₂₉₆Q, Q₃₀₁E,R₃₁₀E,G₃₁₂R,A₃₁₅G
E2	Pa10		TG₁₀₁₆G → TAG(stop codon)
F	Pa27	Premature stop codon	₅₁₁GAA→TAA(stop codon)

Mutational alterations are denoted by single letter codes of amino acids with subscript number denoting its position in protein—the first represents the original while the second letter following the subscripted number denotes the altered one (G- glycine, A–alanine, L- leucine, M- methionine, F- phenylalanine, W- tryptophan, K- lysine, Q- glutamine, E -glutamic Acid, S-serine, P- proline, V-valine, I-isoleucine, Y–tyrosine, R- arginine, N- asparagine, D- aspartic Acid, T–threonine).

Molecular modeling of OprD proteins and mutational analysis

Using homology modeling approach, 3D models of OprD porin from P. aeruginosa PAO1 and their mutant varieties were predicted, refined, and analyzed. The template structure PDB: 1D 4FOZ, chain A with an alignment score of 852, query coverage of 94%, E-value of 0.0, and identity and similarity percentage of 99% was used as the best template for homology modeling of OprD porin from P. aeruginosa PAO1. The structure annotated pairwise alignment and superposition studies between the target and templates showed that RMSD of 2.29 Å with 1507 superimposed backbone atoms. The local and global RMSD between the hypothetical model and the template 4FOZ_A showed RMSD value of 2.52 Å, which is an acceptable cutoff. Furthermore, computational analysis clearly indicated that the predicted models showed good stereochemical validity with more than 90% of the amino acid residues in the most favored region of Ramachandran plot.

Similarly, the force field parameters and energy minimization of the hypothetical models demonstrated reliable results and showed bioactive conformation with minimum energy. Most of the residues in the model were found located in the acceptable regions within the plot predicted by ANOLEA, GROMOS, and VERIFY 3D. The stable secondary structures such as helices, sheets, and coils produced by the amino acids were clearly predicted by the server DSSP. The C-beta interaction energy, all atom pairwise energy, solvation energy, torsion angle energy, secondary structural agreement, and QMEAN score are shown in Supplementary Table S3. Thus, from the computational prediction and analysis, it is clear that the quality of the hypothetical models is reliable and probably acceptable for further studies.

The mutation-induced structural variations observed in the four selected mutants—Pa12, Pa7, Pa25, and Pa27 representing different OprD types were compared with the wild-type reference strain P. aeruginosa PAO1. Pa12 C2-OprD-type mutants were found to harbor several amino acid polymorphisms, while Pa7 symbolized mutants displaying typical deletions/substitutions such as ₃₇₂(VDSSSS -YAGL-)₃₈₃ in addition to amino acid polymorphisms. The remaining two, Pa25 and Pa27, were illustrative of mutants bearing protein truncations at different regions (Fig. 2).

FIG. 2.

(a) Three-dimensional models of porin OprD of the wild-type reference strain, P. aeruginosa PAO1 strain and mutants, Pa12, Pa7, Pa25, and Pa27. Pa12 mutant harbors several amino acid polymorphisms; Pa7 displays the typical ₃₇₂(VDSSSS-YAGL-)₃₈₃ amino acid deletions/substitutions in addition to amino acid polymorphisms; Pa25 mutant truncated protein along with several amino acid changes; Pa27 mutant truncated protein. (b–g) Mutational analysis of the hypothetical models of the OprD porin variants of P. aeruginosa. (b) Pa12, (c) Pa7, and (d) Pa25 OprD protein. Left: side view; Right: Top view looking through the channel. The individual sequences of the OprD porin variants of P. aeruginosa Pa12, Pa7, and Pa25 are given in (e), (f), and (g), respectively, with the mutational variations highlighted in gray.

Restriction mapping of oprD gene

The amplicons of oprD genes obtained from the mutants were found to be about 1,500 bp in size as expected. The gene length for reference strain PAO1 has been reported as 1,332 bp.⁴⁰ In an attempt to further characterize the DNA fragment, the oprD sequence of the reference PAO1 strain retrieved from GenBank and the mutant sequences obtained in the study were together subjected to a scan with the NEB cutter V2.0³² to detect the presence of restriction sites therein. Reproducible restriction patterns were obtained with restriction endonucleases, HincII, PstI, and PvuII. With respect to the wild-type porin gene of PAO1, HincII enzyme was found to restrict the cognate sequence 5′GT(C/T)↓(A/G)AC3′ at 189th and 1308th position, while PstI restricted the sequence 5′CTGCA↓G3′ at 497th and PvuII at 492nd position of the recognition sequence 5′CAG↓CTG3′. The restriction profiles are shown in Supplementary Fig. S1 and the details of the restriction analysis are given in Supplementary Table S4.

Discussion

In P. aeruginosa, OprD porin protein is specialized to facilitate the diffusion of hydrophilic carbapenem antibiotics through the outer membrane into the periplasmic space where they encounter their target, the penicillin-binding protein.⁴¹ Emergence and spread of carbapenem-resistant P. aeruginosa have become a serious concern worldwide. Among different carbapenem resistance mechanisms, such as inducible chromosomal AmpC, reduced antibiotic uptake due to mutant or lost outer membrane OprD porin protein, overexpression of efflux pump, and genetic acquisition of carbapenem hydrolyzing enzymes, downregulation of OprD is known to be the most common.⁴²

In the present study, we have analyzed and listed out mutational variations in ampliconic sequences of porin-oprD and its regulatory gene, mexT, obtained from MDR clinical isolates of P. aeruginosa from Kerala, India. Three-dimensional molecular models of selected mutant OprD-porin proteins were also generated using homology modeling approach to facilitate visualization of the location of mutations and the consequent structural alterations. The oprD sequences were also scrutinized for the presence of RFLPs, if any.

The gene mexT encodes a LysR-type transcriptional activator protein, MexT, which mediates hyperexpression of MexEF-OprN efflux pump in nfxC-type mutants resistant to quinolones. Expression of MexEF-OprN is usually linked with OprD porin downregulation mediated by the regulatory action of the MexT.⁴³ Among 26 P. aeruginosa isolates which displayed downregulation of porin expression, mexT gene-specific amplicons were obtained only in 24 isolates other than Pa2 and Pa6, both of which failed to generate amplicons due to reasons which remain unclear. Similar amplification failures have also been reported earlier by Poonsuk et al.⁴⁴ and recently by us in a related study.¹⁶

It was interesting to note the occurrence of an 8-bp deletion (GGCCAGCC) at nucleotide position 235 of mexT sequences in all isolates. Incidentally, the same deletion at identical location has also been reported previously in different P. aeruginosa strains by many researchers.^45–48 Likewise, Ocampo-Sosa et al.⁴¹ discovered an 8-bp deletion (GCCGGCCA) at position 240; yet, another 8-bp deletion (CGGCCAGC) at 226th nucleotide position has also been reported in other independent studies.^49,50 Notably, isolates such as Pa16 and Pa18 displayed only 99% identity with P. aeruginosa—GenBank accession nos. CP012901.1⁴⁷ and CP025051.1, respectively, compared to complete sequence identity observed with GenBank deposits of other strains. Pa16 showed a novel mutation, ²⁷⁸T→C, while Pa18 was found to harbor another mutation, ¹⁹⁷G→A.

The OprD crystallizes as a monomeric 18-stranded β-barrel comprising 9 loops forming an outer membrane-spanning channel with a positively charged basic ladder—a line of positive charges formed by five arginines and one lysine residue on one side and an electronegative pocket on the other, creating an asymmetric charge distribution along the substrate permeation pathway.¹³ The rates of permeation of basic carbapenems through the OprD porin protein are usually related to their molecular weights (MW). Carbapenems such as imipenem (MW 299) and meropenem (MW 383.5) differ essentially in size and pKa value of their C-2 substituent resulting in a 10-fold increase in the diffusion of imipenem through the channel than that of meropenem.⁵¹ In our study, among isolates with reduced oprD expression, only 53.8% were found to show resistance to imipenem compared to 80.8% resistance against meropenem.

A recent study by Vollan et al.⁵² on in silico structure and sequence analysis of bacterial porins and specific diffusion channels reports occurrence of high mutation rates in sequences encoding surface-exposed loops. It has been suggested that such mutations are likely to contribute toward survival and adaptation mechanisms of the pathogen, including evasion of host immune response. The external loops 2 and 3 of OprD are known to serve as entrance and/or binding sites for specific substrates such as basic amino acids and imipenem within the OprD conduit¹⁰ and mutations therein underlie conformational changes in the protein resulting in imipenem resistance. It is relevant here to point out that in this article, we have referred to the channel protein loops essentially as described by Kos et al.⁵³

Intriguingly, an analysis of the amino acid sequences of C1—four OprD types displaying several polymorphisms (Table 4) revealed that all C2 type isolates bore sensitivity to imipenem despite the presence of similar mutations (T₁₀₃S,K₁₁₅T,F₁₇₀L) in loops, initially designated as L2 and L3 by Epp et al.,⁵⁴ known to be associated with imipenem resistance.^10,42 The fact that deletion of loops 5, 7, and 8 is associated with enhanced susceptibility to beta-lactams, quinolones other than fluoroquinolones, chloramphenicol, and tetracycline reveals that these three loops are also involved in restricting the intracellular accumulation of certain antibiotics. Furthermore, mutations leading to L7 shortening is known to increase meropenem susceptibility.¹⁰

In our study, OprD types D1-D6 showed a stretch of 12 amino acid residues located in loop L8 (formerly L7) replaced by a sequence of 10 amino acid residues within 372nd (VDSSSS-YAGL-) to 383rd position.^7,41,54–58 The shortening of L7 (designated as L8 by us) first described by Epp et al.,⁵⁴ was held responsible for the unusual meropenem hypersusceptibility due to widening of OprD channel opening. Incidentally, we have observed that only a few isolates harboring this OprD alteration ₃₇₂(VDSSSS-YAGL-)₃₈₃ displayed susceptibility to both carbapenems. Thus, carbapenem resistance displayed by most of the isolates belonging to D1-D6 types may perhaps be due to the presence of alternate resistance mechanism(s), which needs further elucidation.

It was contrasting to note that two isolates lacking L7 shortening, Pa6 (A-OprD type) and Pa4 (C1- OprD), still displayed sensitivity to meropenem (Tables 1 and 4). Such unusual meropenem susceptibility of imipenem-resistant clinical strains has been reported earlier by Epp et al.⁵⁴ which may perhaps be related to the role of other porin types (OprF and OprE) involved in meropenem transport.⁵⁹ It may be relevant to mention here that conserved mutations consisting of changes of one hydrophobic residue to another that was also hydrophobic, located in loops connecting the β-sheets or within the β-sheets with side chains pointing outwards of th β-barrel, need not affect the integrity of the porin.⁴¹ In our study, the observation that all three truncated oprD mutant proteins from Pa25, Pa10, and Pa27 displayed resistance to both of the carbapenem antibiotics is as expected.

Sequence analysis of oprD mutants in our study also revealed the occurrence of RFLPs. The loss and gain of restriction sites were clearly evident in the majority of the isolated mutants on restriction mapping of oprD gene sequences using three selected enzymes. The RFLPs discovered in the oprD amplicons of the clinical isolates should prove useful as a potential diagnostic tool for detection of P. aeruginosa porin mutants. Since the oprD gene plays a key role in development of antibiotic resistance, the discovery of RFLP in this study can be exploited to characterize clinical isolates without taking recourse to DNA sequencing in addition to its epidemiological importance.

In conclusion, carbapenem resistance involving OprD-deficient P. aeruginosa can evolve into a serious health threat as a consequence of continued selective pressures due to indiscriminate use of carbapenems.

The present study showed a downregulation of oprD gene in about 90% of the P. aeruginosa clinical isolates with all ampliconic sequences of the regulatory gene mexT displaying an 8 bp (GGCCAGCC) deletion at 235th nucleotide position. Hypothetical models of mutant OprDs enabled clear visualization of substantial portions of the protein lost due to truncation. Overall, among the three modeled mutant proteins, the mutations were predominantly confined to the extracellular loops ranging from 78% in Pa12 to 75% in Pa7 with a noteworthy drop to 57% in Pa25 attributable to truncation of the latter half of the protein at the 708th nucleotide position due to the occurrence of a stop codon. oprD amplicons were also found to display occurrence of RFLPs potentially useful for detection of porin mutants circumventing the need for DNA sequence-based analysis.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Footnotes

Disclosure Statement

All authors declare that they have no conflict of interest.

Funding Information

This study was supported by the Government of India, DST (Department of Science and Technology) - INSPIRE fellowship (Grant No. IF120394) to M.S.

Supplementary Material

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