In silico identiﬁcation of auxiliary genes

required for

-lactam resistance

Volumen 14 Número 1 - 2023

ABSTRACT

In silico idencaon of auxiliary genes required for

β-lactam resistance

(1) Carrera de Nutrición y Dietética. Escuela Superior Politécnica de Chimborazo, Riobamba, 060101, Ecuador.

* Correspondence to: igor.astudillo@espoch.edu.ec

Igor Eduardo Astudillo Skliarova ¹

igor.astudillo@espoch.edu.ec

Staphylococcus aureus is a type of bacteria commonly found on the skin and in the nasal passages of healthy

individuals. However, it can also cause a range of infecons in clinical sengs. One of the most concerning

aspects of S. aureus is its ability to develop anbioc resistance. Methicillin-resistant S. aureus (MRSA) is a strain

of the bacteria that is resistant to many anbiocs and can be dicult to treat. The primary mechanism of

methicillin resistance in MRSA is the presence of the mecA gene, which encodes for a modied penicillin-binding

protein known as PBP2a. This protein has a lower anity for beta-lactam anbiocs. Another gene, blaZ, is

also present in MRSA and encodes for a beta-lactamase enzyme that can hydrolyse and inacvate beta-lactam

anbiocs such as penicillin. In addion, there are several auxiliary factors that can contribute to beta-lactam

resistance. They can include eux pumps, enzymes that modify or degrade anbiocs, and bacterial cell wall

modicaons that reduce the anity of anbiocs for their targets. In this study, with the aid of the in silico

idencaon method, we idenfy the novel auxiliary factors aux1, aux2, aux4, aux11, aux14, aux16 and aux19.

Next, we show that aux2, aux4, aux11, aux14 are not directly involved in β-lactam resistance, but may contribute

through other mechanisms that decrease the ecacy of these anbiocs, whereas aux16 and aux19 are directly

associated with β-lactam and bacitracin resistance, respecvely. Understanding the various auxiliary factors that

contribute to beta-lactam resistance can help guide the development of new anbiocs and other therapeuc

strategies.

Keywords: auxiliary factors, Staphylococcus aureus, β-lactam anbioc, anbioc resistance, in silico

idencaon.

Facultad de

Salud Pública

Facultad de

Salud Pública

CSSN

La Ciencia al Servicio de la Salud y la Nutrición

REVISTA CIENTÍFICA DIGITAL

La Ciencia al Servicio de la Salud y la Nutrición

http://revistas.espoch.edu.ec/index.php/cssn

ISSN 1390-874X

ARTÍCULOS ORIGINALES

1. INTRODUCTION

Staphylococcus aureus is a Gram-posive bacterium

that is commonly found on the skin and mucous

membranes of humans and other animals. It is a

leading cause of healthcare-associated infecons,

including bacteremia, sepsis, toxic shock syndrome,

and skin and so ssue infecons (1). Staphylococcus

aureus is able to acquire resistance to mulple

anbiocs, including methicillin, through the

acquision of anbioc resistance genes. This ability

to resist anbiocs has made it dicult to treat S.

aureus infecons and has led to the emergence

of methicillin-resistant S. aureus (MRSA). MRSA is

highly transmissible and can spread through contact

with infected persons or contaminated surfaces

and, therefore, it poses a major challenge to public

health and medical care (2).

Methicillin-resistant Staphylococcus aureus (MR SA)

is classied into three main types: healthcare-

associated (HA-MRSA), community-associated

(CA-MRSA) and livestock-associated (LA-MRSA)

(3). CA-MRSA strains are usually acquired in the

community and typically causes mild to moderate

skin and so ssue infecons, such as boils

and abscesses. One of the most important and

common CA-MRSA strains is MW2 (4).

MRSA strains possess the mecA gene, which

encodes the alternave penicillin-binding protein

PBP2A. This protein confers resistance to β-lactam

anbiocs by decreasing their ability to bind to

the cell wall. Addionally, some MRSA strains may

encode the beta-lactamase BlaZ, which hydrolyses

β-lactam anbiocs (5).

Although the presence of mecA is necessary

for the resistance to β-lactam anbiocs, it is

not sucient. Therefore, apart from mecA and

blaZ, many other genes involved in resistance to

β-lactam anbiocs have been idened and

Igor Eduardo Astudillo Skliarova

http://revistas.espoch.edu.ec/index.php/cssn

named such as fem genes (factors essenal for

methicillin resistance) and aux genes (auxiliary

genes). Both of these factors are not related to

mecA gene (6). A list of currently known accessory

and auxiliary factors is presented on Table 1.

Auxiliary factors may aect the ability of

anbiocs to penetrate the bacterial cell wall or

alter the expression of resistance genes (28). One

such factor is the presence of eux pumps, which

are membrane proteins that can pump anbiocs

out of the bacterial cell. This can reduce the

concentraon of anbiocs inside the cell and

make it more dicult for the anbiocs to be

eecve (6). Another factor is the presence of

enzymes that can modify or degrade anbiocs.

Table 1: List of auxiliary factors.

For example, some bacteria produce beta-

lactamases that can hydrolyse and inacvate

beta-lactam anbiocs (29). Bacterial cell wall

modicaons, such as the addion of amino acids

or sugars to the pepdoglycan layer, can also

contribute to beta-lactam resistance by reducing

the anity of anbiocs for their targets (30).

Although the mechanisms of resistance to β-lactam

used by S. aureus have been studied for many

years, there are sll many unclear mechanisms.

Therefore, the aim of the present study is to

idenfy novel putave auxiliary factors genes

present in S. aureus using the in silico idencaon

method.

Gene Funcon

glmM

Phosphoglucosamine mutase: conversion of glucosamine-6-phosphate to glucosamine-1-phosphate dur-

ing the early stage of pepdoglycan synthesis (7).

glmS

Glucosamine-fructose-6-phosphate aminotransferase: conversion of D-fructose-6-phosphate to D-glu-

cosamine 6-phosphate (8).

murA Transferase: conversion of UDP-GlcNAc to UDP-GlcNAc-enoylpyruvate (9).

murB Reductase: conversion of UDP-GlcNAc-enoylpyruvate to UDP-MurNAc (9).

murC-F

Mur ligases: sequenal addion of the aminoacids L-Ala, D-Glu, L-Lys, and D-Ala-D-Ala to build the tetra-

pepde side chain (10).

glyS Glycine tRNA synthetase: provides a glycine substrate for the assembly of the pentaglycine bridge (11).

femX Pepdyltransferase: Incorporaon of the rst glycine of the pentaglycine bridge (12).

femA Pepdyltransferase: Incorporaon of the glycines 2 and 3 of the pentaglycine bridge (12).

femB Pepdyltransferase: Incorporaon of the glycines 4 and 5 of the pentaglycine bridge (12).

femC Glutamine synthetase repressor: Parcipates in the tetrapepde side chain amidaon (13).

gatD/murT Amidotransferase complex: amidaon of lipid II (14).

SAV1754 Probably funcons as a lipid II ippase during its translocaon to the outer leaet of the cell membrane (15).

sW Proposed lipid II ippase (16).

pbp1 PBP, possesses transpepdase acvity (17).

pbp2 Bifunconal PBP with transglycosylase and transpepdase domains (17).

pbp4 PBP, possesses transpepdase and carboxipepdase acvies (18).

prsA Probably required for posranscriponal maturaon of PBP2a (19).

fmtA Accessory PBP, possesses transpepdase acvity and has low anity to penicillin (20).

Llm (tarO)

Transferase: transfer of an N-acetylglucosamine-1-phosphate residue from UDP-N-acetylglucosamine to

undecaprenyl-phosphate to produce the WTA intermediate lipid α (21).

tarA Transferase: transfer of an N-acetylmannosamine residue to the lipid α to form the intermediate lipid β (21).

tarB Transferase: transfer of an sn-glycerol-3-phosphate unit to generate the intermediate lipid ϕ.1 (22).

tarD Cydylyltransferase: producon of CDP-glycerol for TarB during WTA synthesis (22).

tarL Cydylyltransferase: required for ribitol phosphate polymerisaon of WTA (22).

tarI CDP-ribitol synthase: Synthesis of CDP-ribitol for TarL during WTA synthesis (22).

tarS

ltaS

Glycosyltransferase: Addion of β-O-GlcNAc to the WTA (22).

LTA synthesis (23).

spsB Signal pepdase enzyme: posranscriponal regulaon of the protein LtaS (24).

pknB Eukaryote-like serine/threonine kinases: Associated with increased resistance to β-lactam anbiocs (25).

sigB Alternave sigma factor: Involved in β-lactam resistance (26).

vraSR

Two-component regulatory system: acvaon of specic genes required for anbioc resistance in re-

sponse to cell wall stress (27).

In silico identiﬁcation of auxiliary genes

required for

-lactam resistance

Volumen 14 Número 1 - 2023

2. METHODOLOGY

In silico gene idencaon

The sequence of the genes of interest was

downloaded from the nucleode databases of the

Naonal Center for Biotechnology Informaon

(NCBI) (hp://www.ncbi.nlm.nih.gov/) and the

Kyoto Encyclopedia of Genes and Genomes

(KEGG) (hps://www.genome.jp/kegg/).

In silico protein characterisaon

With the aid of the programme protein-protein

BLAST (Basic Local Alignment Search Tool) (hp://

blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins),

the translated sequence of the genes was aligned

with the sequence of amino acids of already

known and studied proteins in order to predict

the funcon of the protein encoded by the gene

of interest. Several characteriscs of the protein

such as the isoelectric point (pI), molecular

weight (mW), co-localisaon with other genes,

the transmembrane helices, homology or

structure were predicted with the aid of the

online available programmes: Expert Protein

Analysis System (hps://www.expasy.org/),

DeepTMHMM (hp://www.cbs.dtu.dk/services/

TMHMM/) and Homology detecon & structure

predicon by HMM-HMM comparison (HHpred)

(hps://toolkit.tuebingen.mpg.de/tools/hhpred).

3. RESULTS

In 1999 the idencaon of 21 novel auxiliary

genes was reported in the Microbial Drug

Resistance journal (31). Unfortunately, aer

this, some of these genes have not been further

characterised and the funcon of many of these

auxiliary genes is sll unknown. In this study, we

decided to predict the funcon and properes of

the proteins encoded by uncharacterised genes

by using dierent bioinformacs tools.

The sequence data of the aux genes: aux1,

aux2, aux4, aux11, aux14, aux16 and aux19, was

retrieved with the aid of NCBI using the accession

numbers found in the afore-menoned report.

As these genes were discovered in S. aureus strain

COL. In order to predict the preliminary name

of the gene and the funcon of its product, the

amino acid sequence was aligned with homolog

sequences of other organisms using protein-

protein BLAST. This and other informaon related

to the protein were collected with the aid of the

following online available sowares: ExPaSy,

TMHMM Server v. 2.0 and HHpred. All the data

is shown in Table 2. Our results suggest that,

among all the genes described by De Lencastre

et al. in 1999 (31), aux16 and aux19 were directly

associated with anbioc resistance.

Table 2: Characteriscs of new auxiliary genes generated in the background of S. aureus strain MW2.

ND, not determined; ¹Paral sequence analysis; ²Metallo β-lactamase.

aux

gene

Accession

number

ORF

Putave

gene

Length

(aa)

pI/MW Stoichiometry Solubility Encoded protein Funcon

aux1 Y18639 1 prpC 247 5.10 / 28076 Monomer Soluble

Phosphorylated protein

phosphatase

Cellular regulaon

aux2 Y13639 388 pknB 664 5.76 / 74363 Monomer

Trans-

membrane

Protein kinase Cellular regulaon

aux4 Y18630 - ccpA 329 5.58 / 36060 Monomer Soluble Catabolite Control Protein A Transcriponal regulaon

aux11 Y18632 - lysA 421 5.58 / 47035 Homodimer Soluble

diaminopimelate decar-

boxylase

L-lysine biosynthesis

aux14 Y14324 271 - 303 5.64 / 34812 ND Soluble RNase adapter protein RapZ Cellular regulaon

aux16 AJ131754

cutD 540 8.93 / 60254 Homotrimer

Trans-

membrane

Osmoprotectant transporter

Choline and betaine/carnine

transporter

2 bla

MBL

228 5.68 / 26239 Monomer Soluble MBL

Resistance to broad range of

β-lactam resistance

4 gbsA 496 4.96 / 54623

Homotetra-

mer

Soluble

Betaine aldehyde dehydro-

genase

Glycine, serine and threonine

metabolism. Betaine biosynthesis

via choline pathway

betA 569 7.08 / 63624 Monomer Soluble Choline dehydrogenase

Glycine, serine and threonine

metabolism. Oxidaon of choline

to betaine aldehyde and betaine

aldehyde to glycine betaine

aux19 Y18641

1 vraD 252 7.03 / 27775 Monomer Soluble

Membrane transport and

signal transducon

ABC transporter. Also part of a

two component system. Associa-

ted to bacitracin

vraE

626 9.65 / 70105 Homotrimer

Trans-

membrane

Membrane transport and

signal transducon

ABC transporter. Also part of a

two component system. Associa-

ted to bacitracin resistance