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

Igor Eduardo Astudillo Skliarova

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

This suggests that all the three genes are involved in cellular regulaon. Moreover, a DeepTMHMM

analysis shows that aux2 is a transmembrane protein (Figure 1). The gene aux4 matched the catabolite

control protein a with 99.66% probability (Figure 1), which suggests that this gene is involved in

transcriponal regulaon. Finally, the gene aux11 matched the diaminopimelate decarboxylase gene

with 100% probability (Figure 1). Diaminopimelate decarboxylase is involved in L-lysine biosynthesis

(32).

The DNA sequences aux1, aux2, aux4 and aux14 are associated with regulaon mechanisms,

whereas aux11 is associated with biosynthec pathways.

The genes aux1, aux2 and aux14 matched respecvely the phosphorylated protein phosphatase with

100% probability, the protein kinase with 100% probability and RNase adapter protein RapZ with 100%

probability (Figure 1).

Figure 1: HHpred analysis of putave auxiliary factors aux1 (a), aux2 (b), aux4 (c), aux11 (d) and aux14 (e), and DeepTMHMM

analysis of aux2, showing the presence of a membrane-bound domain.

In silico identiﬁcation of auxiliary genes

required for

-lactam resistance

Volumen 14 Número 1 - 2023

The DNA sequence aux19 was not directly associated with β-lactam resistance, but was associated

with bacitracin resistance.

The DNA sequence of aux19 comprises two ORFs. These ORFs were predicted to act as ABC transporters

and be part of a two component system (Table 2)(Figure 3). Although these ORFs were not predicted

Figure 2: HHpred analysis of the putave proteins associated with the putave auxiliary factor aux16: cutD (a), blaMBL

(b), gbsA (c) and betA (d), and DeepTMHMM analysis of putave protein cutD, showing the presence of membrane-bound

domains.

The DNA sequence aux16 could be associated with β-lactam resistance

The DNA sequence aux16 comprises 5 open reading frames (ORFs). Among these ORFs, the ORF2

was predicted with 99.85% probability to belong to the Subclass B3 metallo-beta-lactamases (MBL)

(Figure 2), which are a type of bacterial enzymes that can inacvate many beta-lactam anbiocs,

such as penicillins, cephalosporins, and carbapenems (33). Addionally, other ORFs associated with

aux16 may be involved in glycine, serine and threonine metabolism, betaine biosynthesis via choline

pathway and oxidaon of choline to betaine aldehyde and betaine aldehyde to glycine betaine (Table

2). Interesngly, the ORF1 of aux16 was predicted to be a membrane-anchored protein (Figure 2) and

act as a Choline and betaine/carnine transporter.

Igor Eduardo Astudillo Skliarova

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

4. DISCUSSIONS

As already menoned, auxiliary factors

signicantly contribute to β-lactam resistance

and during the last decades many of them have

been described. These factors have been studied

with the aid of transposon mutagenesis using

the transposon (Tn551) (35). In 1994, a large

Tn551 library produced in the background of S.

aureus strain COL was screened for mutants with

decreased levels of resistance to methicillin. The

complete DNA sequence of the 21 new auxiliary

genes derived from this library was published in

1999 (31).

to be directly associated with β-lactam resistance (Table 2)(Figure 3), they were predicted to be

associated with resistance to bacitracin, which is an anbioc that inhibits the synthesis of the cell

wall (34).

The DNA sequence aux19 was not directly associated with β-lactam resistance, but was associated

with bacitracin resistance.

The DNA sequence of aux19 comprises two ORFs. These ORFs were predicted to act as ABC transporters

and be part of a two component system (Table 2)(Figure 3). Although these ORFs were not predicted

to be directly associated with β-lactam resistance (Table 2)(Figure 3), they were predicted to be

associated with resistance to bacitracin, which is an anbioc that inhibits the synthesis of the cell

wall (34).

a b

Figure 3: HHpred analysis of the putave proteins associated with the putave auxiliary factor aux19: vraD (a) and vraE (b),

and DeepTMHMM analysis of putave protein vraE, showing the presence of membrane-bound domains.

In this study, seven of the 21 new auxiliary genes

were characterised by in silico sequence analysis.

As we can observe in Table 9, most of the genes

are involved in metabolic pathways, transport

and regulaon of gene expression.

It is known that any kind of selecve pressure,

including anbioc exposure, causes dramac

changes within the cell and this implies addional

metabolic costs. Therefore, in order to withstand

the damage caused by cell wall targeng

anbiocs, the cell must produce addional

In silico identiﬁcation of auxiliary genes

required for

-lactam resistance

Volumen 14 Número 1 - 2023

proteins required for cell wall maintenance (36).

Notably, one of the aux genes – aux11 has high

homology with the gene lysA, which encodes

diaminopimelate decarboxylase. This protein

is required for the synthesis of the amino acid

L-lysine (32), which is part of the tetrapepde

side chain of the PG (37). In consequence, it is

possible to speculate that, in the presence of

β-lactam anbiocs, enhanced expression of

diaminopimelate decarboxylase is crucial to

the cross-linking of PG, performed by penicillin

binding proteins.

When resistant bacteria are exposed to anbiocs,

before they synthesise anbioc resistance

proteins such as PBP2a, they temporarily become

more vulnerable to osmoc stress. Therefore, the

bacterial cell must acvate genes required for

the transport or synthesis of osmoprotectants

or compable solutes, which contribute to

withstanding osmoc stress (38). Some putave

genes that might play that important role have

been idened in this study: cutD, gbsA and

betA. All these genes were inially idened as

aux16. The putave gene cutD was predicted to

have transmembrane domains, which correlates

with the predicted funcon of its product. The

gene cutD may be required for the transport of

osmoprectants such as betaine or precursors

of other osmoprotectants such as choline (39).

The putave genes gbsA and betA encode the

proteins: betaine aldehyde dehydrogenase

and choline dehydrogenase, respecvely.

Betaine aldehyde dehydrogenase mediates the

biosynthesis of the osmoprotectant betaine,

whereas choline dehydrogenase is responsible

for the synthesis of the osmoprotectant glycine

betaine (40).

β-lactam anbiocs induce the expression of

operons that contain β-lactam resistance genes.

Furthermore, there is evidence that β-lactam

anbiocs also trigger a global stress response in

bacterial cells. This occurs when the cell detects

cell-wall synthesis perturbaons with the aid of

the specic VraS/R two-component system (27).

In this study we idened two putave genes:

vraD and vraE (inially idened as aux19), whose

products might be part of VraS/R two-component

system. The acvaon of this two-component

system leads to massive overexpression of genes,

many of which are crucial for β-lactam anbioc

resistance (27). For instance, VraS/R induces the

expression of RelP and RelQ, which synthesises

alarmones during the exponenal phase of

growth (41). Alarmones are generally produced

during nutrient starvaon (for instance, amino

acid starvaon) and play a principal role in

stringent response, whereby the synthesis of

rRNA, tRNA and amino acids available in the

cell is repressed and synthesis of amino acids

present in insucient amounts within the cell

is induced (42). The principal known alarmones

are guanosine tetraphosphate (ppGpp) and

guanosine pentaphosphate (pppGpp). During

nutrient starvaon, these alarmones are

produced by specic proteins such as RelA or

SpoT, which become acvated when uncharged

tRNA molecules bind the ribosomal site A (43).

There is evidence that this alarmones increase

the resistance of MRSA to anbiocs (44).

The global stress response requires the

parcipaon of many dierent genes involved

in cascade reacons such as protein kinases,

phosphatases or transcriponal regulators.

These molecules play similar roles in prokaryotes

and eukaryotes (45). We idened three putave

genes whose products may parcipate in this

cascade: prpC, pknB and ccpA (inially idened

as aux1, aux2 and aux4, respecvely). We can

presume that the products of these genes

may also be involved in the cascade reacons

triggered by the VraS/R two-component system,

but this proposed linkage must be veried

experimentally.

5. CONCLUSIONS

In conclusion, we can conrm that the gene

blrA is required for β-lactam resistance in the

CA-MRSA strain MW2, but does not aect the

expression of the gene mecA or the structure of

the wall teichoic acids. At the same me, many

auxiliary genes present in CA-MRSA strain MW2

may be involved in global cellular regulaon,

stress response and metabolic pathways.

The author declares no conict of interest

The author takes complete responsibility for the

informaon presented in this original scienc

arcle

6. CONFLICT OF INTEREST

7. LIMITATIONS OF LIABILITY

The present Research Project was funded by

Secretaría Nacional de Educación Superior,

Ciencia, Tecnología e Innovación (SENESCYT) in

2016.

8. FUNDING

Igor Eduardo Astudillo Skliarova

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

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I would like to thank my former MSc supervisor

Dr. Guoqing Xia for providing feedback on the

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