Conserved sequences of genes coding for the multidrug resistance pump AcrAB-TolC of Escherichia coli suggest their involvement into permanent cell “cleaning”

Karakozova M.V.; Nazarov P.A.

CONSERVED SEQUENCES OF GENES CODING FOR THE MULTIDRUG RESISTANCE PUMP ACRAB-TOLC OF ESCHERICHIA COLISUGGEST THEIR INVOLVEMENT INTO PERMANENT CELL "CLEANING"

Karakozova MV1 Nazarov PA2

1 Research Centre of Medical Genetics, Moscow

2 Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow

Multidrug resistance pumps (MDR pumps) of bacteria confer protection against aggressive environmental factors. The genes coding for MDR pumps are thought to be variable. They belong to the group of the so-called contingency genes, i.e are necessary for bacterial adaptation to the changing environment. The aim of the present work was to establish how conserved are the sequences of genes coding for MDR pumps. We analyzed the sequences of AcrA, AcrB and TolC proteins of different Escherichia coli strains. Using sequence alignment tools, we demonstrated that strains originating in different countries and cultured in the labs for a long time are amazingly conserved in terms of AcrAB-TolC sequences. They resemble housekeeping genes, suggesting the involvement of the AcrAB-TolC pump into permanent "cleaning" of various biotic and abiotic agents.

Keywords: multidrug resistance, antibiotic, AcrAB-TolC, sequence alignment, Escherichia coli, pump, transporter, biocide, contingency genes, housekeeping genes

Acknowledgement: the authors wish to thank students (Dzhafarova T, Mamedova D, Maushev F, Miranian K, Omarova D and Rodionova E) and the Department of Morphology and Pathology of Medical Institute REAVIZ for the provided data, their analysis and discussion.

Correspondence should be addressed: Marina Karakozova

Microrayon 1, d.15, kv. 43, Zaraisk, Zaraiski r-n, Moscow region, 140601; [email protected] Received: 19.02.2018 Accepted: 30.03.2018 DOI: 10.24075/brsmu.2018.024

КОНСЕРВАТИВНОСТЬ ПОСЛЕДОВАТЕЛЬНОСТЕЙ ГЕНОВ ПОМПЫ МНОЖЕСТВЕННОЙ ЛЕКАРСТВЕННОЙ УСТОЙЧИВОСТИ ACRAB-TOLC ESCHERICHIA COLI КАК ПРИЗНАК ВОВЛЕЧЕННОСТИ В ПЕРМАНЕНТНУЮ «УБОРКУ» БАКТЕРИАЛЬНОЙ КЛЕТКИ

М. В. Каракозова1 П. А. Назаров2

1 Медико-генетический научный центр, Москва

2 Научно-исследовательский институт физико-химической биологии имени А. Н. Белозерского, Московский государственный университет имени М. В. Ломоносова, Москва

Помпы множественной лекарственной устойчивости (МЛУ) помогают бактериям защищаться от неблагоприятного воздействия окружающей среды. Считается, что гены, кодирующие помпы МЛУ, вариабельны и относятся к так называемым генам «роскоши», т. е. предназначены для адаптации бактерий к изменению окружающих условий. Целью работы было проверить насколько консервативны последовательности генов помпы МЛУ. Для этого проводили анализ последовательностей белков AcrA, AcrB и TolC для различных лабораторных штаммов Escherichia coli. Методом выравнивания последовательностей было показано, что штаммы из разных стран, культивируемые в лабораториях уже долгое время, имеют удивительную консервативность последовательностей белков помпы AcrAB-TolC. Она напоминает консервативность генов «домашнего хозяйства», что, по-видимому, говорит о вовлеченности помпы МЛУ AcrAB-TolC в перманентную «уборку» клетки от различных веществ биотического и абиотического происхождения.

Ключевые слова: множественная лекарственная устойчивость, антибиотик, AcrAB-TolC, выравнивание последовательностей, Escherichia coli, помпа, транспортер, биоцид, гены «роскоши», гены «домашнего хозяйства»

Благодарности: авторы благодарят студентов (Джафарову Т., Мамедову Д., Маушева Ф., Миранян К., Омарову Д. и Родионову Э.) и сотрудников кафедры морфологии и патологии Московского медицинского университета «Реавиз» за предварительные данные, их анализ и дискуссию.

gg Для корреспонденции: Марина Викторовна Каракозова

Микрорайон 1-й, д. 15, кв. 43, г. Зарайск, Зарайский р-н, Московская обл., 140601; [email protected]

Статья получена: 19.02.2018 Статья принята к печати: 30.03.2018

DOI: 10.24075/vrgmu.2018.024

The gram-negative gammaproteobacterium Escherichia coli (E. coli) was first discovered by Theodor Escherich in the stool samples of healthy individuals in 1885 [1]. E. coli naturally inhabits the lower intestines of warm-blooded species and is an important object of research. Four strains of E. coli, including K-12, B, W and C, are now used as model organisms. Strain K-12 was first isolated at Stanford university in 1922 [2]. Strain B was described by d'Herelle at the Pasteur Institute in Paris

in 1918 [3]. The other 2 strains are less common. Strain C was discovered by Margaret Lieb in 1951 [4, 5], and strain W was originally reported by Selman Waksman in 1943 [6]. Strains comprising groups K-12 and B are the most widespread and best known. Laboratory strains have "evolved" to lose some of their properties, such as the ability to form biofilms on abiotic surfaces, and therefore can be advantageously used in research studies, especially for the discovery of novel antibiotics [7].

The pressure of both natural and artificial selection existing in laboratories has produced numerous derivatives of K-12 and B that are now used all over the world (Table 1). Among the derivatives of strain B are BL21 and bl21(de3); DH5a, JM109, W3110, XL-1 Blue, and MG1655 are examples of strain K-12 derivatives.

Discovery of novel antibiotics or their effective alternatives is a pressing challenge. One of the most promising areas of research is identification of multidrug resistance (MDR) pump inhibitors. MDR pumps are responsible for removing antibiotics from the bacterial cell. Studies of deletion mutants with knocked-out genes coding for MDR pumps demonstrate that minimum effective inhibitory concentrations of antibiotics in their case are several times lower than usual [8]. This may help to reduce both treatment costs and the toxic effect of antibiotic therapies on the patient. Although effects of MDR pumps on antibacterial agents are actively studied, there is an extensive list of objective factors preventing cross-study comparisons, such as different genetic backgrounds of the strains. Even for such closely related strains as W3110 and MG1655 [9], the number of differences at genomic sites can be over 200, impeding comparison. Because bacterial resistance to drugs depends on the presence or absence of efflux pumps, we hypothesized that E. coli strains with identical sequences of MDR pumps might have comparable or equal resistance. To check this supposition, we selected the AcrAB-TolC pump. We aimed to compare sequences of AcrA, AcrB and TolC proteins obtained from different laboratory strains of E. coli and to study the associations between drug resistance and possible mutations if such were present in a sequence.

METHODS

Selecting an object

For our study we selected a few K-12 strains: W3110, MG1655, NEB 5-alpha, MDS42, GM4792, AG100, MC4100, DH10B, ER3413, HMS174, BW2952, and BW25113, as well as strain BL21(DE3) from group B. Their acrA, acrB and tolC sequences are known and stored in databases (Table 2).

Selecting a reference sequence

When selecting a reference sequence, we bore in mind a large number of deletion mutants in E. coli K-12 BW25113. It is a parent strain for the Keio collection, which comprises E. coli

Table 1. Geographic origin of E. coli strains used in this work

strains with 3,985 deletions (of 4,288 total E. coli genes) [10]. Sequence AIN30961.1 was selected as a reference sequence for AcrA; AIN30960.1, as a reference sequence for AcrB, and AIN33386.1, as a reference sequence for TolC.

Sequence alignment

Sequences were analyzed using a standard local alignment tool NCBI BLASTp, which allows comparison of multiple alignments [11], and the STRING database [12]. Visual representation of the results was done in NCBI MSA Viewer [13]. Each protein sequence was aligned against its reference sequence.

RESULTS

It is known that bacterial resistance can be a product of: 1) accumulation of resistance genes in plasmids; 2) increased expression of genes coding for MDR pumps; 3) gene duplication; 4) accumulation of mutations [14, 15]. Increased expression and accumulation of mutations in the genes coding for MDR pumps can result in single nucleotide polymorphisms (SNPs) in the amino acid sequences of proteins. Therefore, bacterial resistance can be predicted by sequence analysis.

Bacterial genes are subdivided into housekeeping genes, which support vital functions of the cell, and contingency genes, which play an important role in bacterial adaptation to the changing environment. Housekeeping genes usually have a low mutation rate, while contingency genes tend to demonstrate a high mutation rate [16]. It is believed that genes coding for multidrug efflux pumps are contingency genes; therefore, the proteins they encode are expected to have variable primary structures. Because laboratory strains are usually subject to the pressure of natural selection induced by various biocides and mutagens, the strains that have been cultured in world laboratories for over 100 years, as well as their derivatives, might be different in terms of their amino acid polymorphisms. The strains compared in this work originate from different countries and continents (Table 1), so we can infer the presence of mutations in one of the AcrAB-TolC-encoding genes.

However, the analysis of aligned sequences of AcrA (Fig. 1), AcrB (Fig. 2) and TolC (Fig. 3) proteins (substrain BW25113), those of strain K-12 (substrains W3110, MG1655, NEB 5-alpha, MDS42, GM4792, AG100, MC4100, DH10B,

Strain Institution City, country

MG1655 University of Wisconsin Milwaukee, USA

W3110 Nara Institute of Science and Technology Ikoma, Japan

BL21(DE3) Korea Research Institute of Bioscience and Biotechnology Daejeon, South Korea

MDS42 Osaka University Osaka, Japan

MC4100 University of Kiel, Germany Kiel, Germany

BW25113 Universite de Sherbrooke, Canada Sherbrooke, Canada

ER3413 New England Biolabs Ipswich, USA

AG100 University of Exeter Exeter, UK

NEB 5-alpha New England Biolabs Ipswich, USA

HMS174 Austrian Centre of Industrial Biotechnology Graz, Austria

BW2952 Nankai University Nankai, China

DH10B University of Wisconsin-Madison Madison, USA

GM4792 Beijing Normal University Beijing, China

Table 2. Accession numbers for the stored protein sequences of acrA, acrB and tolC genes

Substrain Strain AcrA AcrB TolC

MG1655 K-12 NP_414996.1 NP_414995.1 NP_417507.2

W3110 K-12 BAE76242.1 BAE76241.1 BAE77091.1

NEB 5-alpha K-12 AOO68785.1 AOO68784.1 AOO71261.1

MDS42 K-12 BAL37669.1 BAL37668.1 BAL39694.1

GM4792 K-12 AKK16793.1 AKK13611.1 AKK18828.1

AG100 K-12 CQR80062.1 CQR80061.1 CQR82466.1

MC4100 K-12 CDJ70932.1 CDJ70931.1 CDJ73817.1

DH10B K-12 ACB01590.1 ACB01589.1 ACB04120.1

ER3413 K-12 AIZ54314.1 AIZ54313.1 AIZ52829.1

HMS174 K-12 CDY55568.1 CDY55565.1 CDY61615.1

BW2952 K-12 ACR63806.1 ACR63808.1 ACR65687.1

BW25113 K-12 AIN30961.1 AIN30960.1 AIN33386.1

BL21(DE3) B ACT42313.1 ACT42312.1 ACT44711.1

ER3413, HMS174, and BW2952) and those of strain B (substrain BL21(DE3)) reveals the absence of polymorphisms in all three proteins constituting the AcrAB-TolC efflux pump, regardless of whether the strain belongs to the derivatives of K-12 or B.

Considering the fact that E. coli mutation rate is ~1*10-3 per genome per generation [17] or even higher (3-4*10-3 per genome per generation) [18], we hypothesize that the AcrAB-TolC pump sequence is conserved. Given the same sequence coverage for all studied proteins (397 amino acid residues for AcrA, 1049 amino acid residues for AcrB and 493 amino acid residues for TolC), the sequence identity was 100%.

DISCUSSION

According to the currently existing classification, strains from group B and K-12 belong to phylogroup A [19], which may explain the similarity of amino acid sequences between all three proteins but not their identity. Our findings allow us to conclude the presence of a consensus sequence of a highly conserved AcrAB-TolC ensemble. Thus, the selected protein reference sequences AcrA (AIN30961.1 for AcrA, AIN30960.1 for AcrB and AIN33386.1 for TolC, respectively) are consensus for the studied E. coli strains.

Fig. 1. Alignment of AcrA sequences for strains K-12 and B against the reference AcrA sequence of substrain BW25113

Sequence ID Start | MM 200 300 400 500 600 700 £ 0 900 10001049 End Organism

AIN3096CI.1 V 1 Escherichia coli BW25113

ACT42312.1 s 1 Eschen dl is coli BL21CDE3)

ACR63eoe.i b 1 Escherichia coli BW2952

cdy35565.1 b 1 Escherichia coli

A1Z54313.1 s 1 Escherichia coli K-12

ACB015B9.1 b 1 Escherichia coli str. K-12 s..

CDJ70931.1 b 1 Escherichia coli str, K-12 s..

COR8OO6I.i s 1 Escherichia coli K-12

AKK13611.1 b 1 Escherichia coli K-12

E3AL37663.1 b 1 Escherichia coli str, K-12 s..

A0068784.1 s 1 Escherichia coli

BAE76241.1 b 1 Escherichia coli str. K-12 s..

NP 414995.1 b 1 Escherichia coli str, K-12 s

Fig. 2. Alignment of AcrB sequences for strains K-12 and B against the reference AcrB sequence of substrain BW25113

Sequence ID Start 1 50 100 150 200 250 300 350 400 450 493 End Organism

consensus

AIN333861 b Escherichia coli BW25113

ACT44711.1 b Escherichia coli BI21(0E3)

ACR65687.1 b Escherichia coll EW2952

CDY61615.1 b Escherichia coli

AIZ52829.1 b Escherichia coli K-12

ACB04120.1 b Escherichia coll str. K-12 s

CDJ73817.1 b Escherichia coli str. K-12 s

COK82466.1 b Escherichia coli K-12

AKK1B82S.1 b Escherichia coli K-12

BA1396S4.1 b Escherichia coll str. K-12 s

A0071261.1 b Escherichia coli

BAE77091.1 b Escherichia coli str. K-12 s

INP 417507.2 b Escherichia coli str. K-12 s.

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Fig. 3. Alignment of TolC sequences for strains K-12 and B against the reference TolC sequence of substrain BW25113

The discovered sequences are consensus for all representatives of group A and possibly other phylogroups, including B1, B2, D, and E, which can facilitate normalization of sequences against their consensus counterparts.

The absence of point mutations in the genes coding for protein components of the AcrAB-TolC pump in all studied strains is indicative of the strict selection control, as is the case with housekeeping genes. Such control is particularly important for the major multidrug efflux pump of E. coli (AcrAB-TolC) responsible for removing benzalkonium chloride, ethidium bromide, indole, hexane, antibiotics (erythromycin, ciprofloxacin, etc.), rhodamine, berberine and also triphenylphosphonium and its derivatives from the cell [20-21].

It would be wrong to see genes coding for MDR pumps as responsible for biocide resistance only. They have a role in bacterial colonization and persistence [22], so it is not limited to

defense against antibiotics. It appears that proteins produced by MDR pump-encoding genes routinely protect bacterial cells from various biotic and abiotic agents and can be regarded as housekeeping genes engaged in permanent cell "cleaning", unlike contingency genes that get involved only at certain times.

CONCLUSION

Our findings suggest a unique role of the AcrAB-TolC multidrug resistance pump in E. coli. The protein sequence of AcrAB-TolC has turned to be surprisingly conserved. This provides a fresh look at AcrAB-TolC from a different angle: this pump ensures permanent protection against aggressive environment, determines bacterial resistance to antibiotics or their alternatives and even ensures bacterial survival.

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Литература

1. Escherich T. Die Darmbakterien des Neugeborenen und Säuglinge. Fortschr. Med. 1885; 3: 515-522.

2. Daegelen P, Studier FW, Lenski RE, Cure S, Kim JF. Tracing ancestors and relatives of Escherichia coli B, and the derivation of B strains REL606 and BL21(DE3). J Mol Biol. 2009; 394 (4): 634-43.

3. Bachmann BJ. Pedigrees of Some Mutant Strains of Escherichia coli K-12. Bacteriological Reviews, 1972; 36 (4): 525-557.

4. Lieb M, Weigle JJ, Kellenberger E. A study of hybrids between

two strains of E. coli. Journal of Bacteriology. 1955; 69 (4): 468-471.

5. Lieb M. Forward and Reverse Mutation in a Histidine-Requiring Strain of Escherichia Coli Genetics. 1951; 36 (5): 460-477.

6. Archer CT, Kim JF, Jeong H, Park JH, Vickers CE, Lee SY, et al. The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli. BMC Genomics. 2011; 12: 9.

7. Vidal O, Longin R, Prigent-Combaret C, Dorel C, Hooreman M, Lejeune P. Isolation of an Escherichia coli K-12 mutant strain able

to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression. J Bacteriol. 1998; 180 (9): 2442-2449.