antibiotics

Article
Comparative Transcriptome Analysis Reveals Differentially
Expressed Genes Related to Antimicrobial Properties of
Lysostaphin in Staphylococcus aureus
Xianghe Yan 1, * , Yanping Xie 2 , Charles Li 3 , David M. Donovan 3 , Andrew Gehring 2 , Peter Irwin 2
and Yiping He 2, *

1 Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center,
Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA
2 Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center,
Agricultural Research Service, United States Department of Agriculture, Wyndmoor, PA 19038, USA;
[email protected] (Y.X.); [email protected] (A.G.); [email protected] (P.I.)
3 Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center,
Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA;
[email protected] (C.L.); [email protected] (D.M.D.)
* Correspondence: [email protected] (X.Y.); [email protected] (Y.H.); Tel.: +1-301-504-9292 (X.Y.);
+1-215-233-6422 (Y.H.); Fax: 1-301-504-8623 (X.Y.); 1-215-233-6581 (Y.H.)

Abstract: Comparative transcriptome analysis and de novo short-read assembly of S. aureus Newman
strains revealed significant transcriptional changes in response to the exposure to triple-acting



staphylolytic peptidoglycan hydrolase (PGH) 1801. Most altered transcriptions were associated with


the membrane, cell wall, and related genes, including amidase, peptidase, holin, and phospholipase
Citation: Yan, X.; Xie, Y.; Li, C.;
Donovan, D.M.; Gehring, A.; Irwin,
D/transphosphatidylase. The differential expression of genes obtained from RNA-seq was confirmed
P.; He, Y. Comparative Transcriptome by reverse transcription quantitative PCR. Moreover, some of these gene expression changes were
Analysis Reveals Differentially consistent with the observed structural perturbations at the DNA and RNA levels. These structural
Expressed Genes Related to changes in the genes encoding membrane/cell surface proteins and altered gene expressions are
Antimicrobial Properties of the candidates for resistance to these novel antimicrobials. The findings in this study could provide
Lysostaphin in Staphylococcus aureus. insight into the design of new antimicrobial agents.
Antibiotics 2022, 11, 125. https://
doi.org/10.3390/antibiotics11020125 Keywords: Staphylococcus aureus; lysostaphin; peptidoglycan hydrolase (PGH); genomics; gene expression;
Academic Editors: RNA-seq
Daniele Castagnolo, Jürgen Brem,
Sónia Silva and Mark G. Moloney

Received: 14 December 2021

1. Introduction
Accepted: 11 January 2022
Published: 18 January 2022
Lysostaphin [1], classified as a prototype III bacteriocin, is a glycyl-glycine bacteriocin
peptidoglycan hydrolase (PGH) secreted by Staphylococcus simulans. PGH is known to
Publisher’s Note: MDPI stays neutral
degrade the peptidoglycans in Staphylococcus aureus cell walls, resulting in cell lysis [2].
with regard to jurisdictional claims in
The antimicrobial properties of lysostaphin have been demonstrated in vitro and in vivo [3].
published maps and institutional affil-
Several mechanisms of resistance to lysostaphin have been proposed: for example, re-
iations.
duced S. aureus fitness [4]; mutations in lysostaphin-resistant S. aureus femA [5]; replacing
the Gly3 of the pentaglycine ligand by serine in the peptidoglycan pentaglycine cross
bridge or a shortened cross-bridge [6–10]; alterations in the plasmid-borne lss (lysostaphin
Copyright: © 2022 by the authors.
endopeptidase) and lif (pACK1) genes [9,11].
Licensee MDPI, Basel, Switzerland. In this study, we have identified the genetic mechanisms of resistance to peptidoglycan
This article is an open access article hydrolases via repeated exposure of S. aureus Newman_2010 strain (cultured wild-type
distributed under the terms and (WT) S. aureus Newman in the year of 2010) to sublethal concentrations of a genetically
conditions of the Creative Commons engineered, triple-acting, staphylolytic, peptidoglycan hydrolase (PGH1801) [2,12]. The
Attribution (CC BY) license (https:// resultant mutant strain S. aureus Newman 1801_2010 has resistance to lysostaphin with a
creativecommons.org/licenses/by/ >2-fold increase in minimum inhibitory concentration (MIC) relative to the wild-type strain
4.0/). Newman_2010 without exposure to any PGH [2,13–15]. This phenotype does not appear to

Antibiotics 2022, 11, 125. https://doi.org/10.3390/antibiotics11020125 https://www.mdpi.com/journal/antibiotics

Antibiotics 2022, 11, 125 2 of 13

be caused by the resistance mechanisms described previously because no changes were

found in the known resistance genes (data not shown).
Environmental conditions experienced by multiple generations of S. aureus New-
man_2010 cultured with PGH1801 could alter this organism’s differential survival and re-
production characteristics. In response to sublethal concentrations of triple-acting PGH1801,
S. aureus Newman could produce a unique phenotypic adaptation. This property is known
as phenotypic plasticity [16]. Such ‘transgenerational’ plasticity could provide a competitive
advantage in growth resulting in long-term environmental variation [17–20].
Next-generation sequencing allows the study of any organism at the genomic and
transcriptomic levels. Exploring whole-genome expression using next-generation sequenc-
ing and RNA-seq provides a more comprehensive understanding than just looking at DNA
primary structure, since RNA-seq captures actively transcribed regions and, therefore, can
ascertain the molecular basis of a phenotype [21–25]. In this study, we have applied DNA-
and RNA-seq analyses by comparing genome-wide single nucleotide polymorphism (SNP)
and insertion/deletion (InDel) results, as well as by measuring the differential expression
levels of thousands of genes simultaneously between the strains of wild-type Newman_2010
and mutant 1801_2010 to identify mutations associated with the above resistant pheno-
types. Meanwhile, our comparative transcriptome analysis and de novo short-read assembly
revealed the transcriptional changes of S. aureus in response to lysostaphin treatment. All
the pooled RNA-seq data were used to compare and establish genome-wide SNP and
InDel results between the mutant 1801_2010 and WT Newman_2010. Reverse transcription
qPCR was used to confirm the RNA-seq results. Computational analyses, including gene
ontology and KEGG pathway enrichment, were also employed. We aim to identify the
mutations which provide resistance to the triple-acting PGH. The findings in this work
could provide insights into the design of new antimicrobial agents.

2. Materials and Methods

2.1. Bacterial Strains, Culture Conditions and Mutant Isolation
S. aureus strain Newman_2010 (wild-type) was used in this study [2]. A mutant strain
1801_2010 was developed by repeated exposure of the wild-type strain to sublethal concen-
trations of the triple-acting PGH1801, a fusion protein containing 3 active domains [2,12,14].
Briefly, S. aureus Newman 2010 bacteria were incubated with two-fold serial dilutions of
PGH1801 (10, 5, 2.5, 1.25, and 0.63 µg/well). On day 1 of the MIC assay, bacteria growing in
the first turbid well (a sublethal concentration) next to clear wells (lethal concentration) were
selected as an inoculate for next MIC assay. Over this time of repeated exposures, bacteria
developed more and more resistance to PGH1801. After 10 cycles of exposure of S. aureus
Newman_2010 to sublethal concentrations of PGH1801, a mutant strain (1801_2010) with
a 2- fold increase in MIC was isolated. Both wild-type and mutant S. aureus strains were
grown at 37 ◦ C on tryptic soy agar (TSA) or in tryptic soy broth (TSB). Growth curves of
the strains were determined and shown in Supplementary Figure S1. The wild-type and
mutant strains displayed a similar growth pattern with the doubling times 47.9 ± 0.431 and
48.1 ± 0.458 min, respectively.

2.2. RNA Preparation

S. aureus Newman_2010 and 1801_2010 cultures were grown in triplicates to around
0.4 of OD600, and harvested by centrifugation at 4000× g for 15 min at 4 ◦ C. Bacterial
pellets were then immediately resuspended into TRI Reagent (Molecular Research Center,
Inc., Cincinnati, OH, USA). Total RNA was extracted using Direct-Zol RNA MiniPrep
according to the manufacturer’s instructions (Zymo Research, Irvine, CA, USA). Each
RNA sample (50 µg) was treated with 5 U of RNase-free DNase I in the presence of 80 U
of RNasin (Promega) at 37 ◦ C for 2 h to remove traces of chromosomal DNA, and then
purified using an RNA Clean and Concentrator™-25 kit (Zymo Research, Irvine, CA, USA).
Eluted RNAs were quantified using a Qubit 2.0 fluorometer with a Qubit RNA HS Assay
Kit (Life Technologies, Carlsbad, CA, USA). Quality of each RNA sample was evaluated
Antibiotics 2022, 11, 125 3 of 13

using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) with an RNA
6000 Nano kit. All the samples showed the RNA integrity numbers (RIN) above 8. The
efficiency of DNase I treatment of the RNA samples was assessed by PCR amplification of a
S. aureus house-keeping gene (gyrA) with a positive (S. aureus genomic DNA) and negative
control (nuclease-free water).

2.3. RNA-seq Library Construction and Sequencing

Prior to RNA-seq library preparation, ribosomal RNA was depleted from the total RNA us-
ing a Ribo-Zero rRNA Removal Kit for Gram-positive Bacteria (Illumina, San Diego, CA, USA)
according to the manufacturer’s instructions. A Qubit RNA HS Assay and Agilent Bioanalyzer
with an RNA 6000 Pico kit were used to assess the quantity and quality of the rRNA-depleted
RNA samples. Six libraries (2 strains × 3 biological replicates) were constructed using a TruSeq
Stranded mRNA Sample Preparation kit (Illumina, San Diego, CA, USA) following the man-
ufacturer’s recommendations. The libraries were sequenced on an Illumina Miseq platform
using 300 base- length read chemistry in a paired-end mode.

2.4. Reverse Transcription Quantitative PCR (RT-qPCR) Analysis

RT-qPCR was performed on selected genes based on the relative transcription levels
(logFC > 2.0) obtained from RNA-seq results. Primers were designed using the NCBI primer
designing tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi) (accessed on
10 January 2022). Reverse transcription reactions were carried out using random primers
and SuperScript II Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA). Quan-
tification of cDNA was performed on a 7500 real-time PCR system (Applied Biosystems,
Foster City, CA, USA) using a SYBR®® Green PCR Master Mix. The gyrA gene was used
as a reference for data normalization. Housekeeping genes gmk and pta were included
as controls to ensure data reliability. All the samples were analyzed in three biological
and technique triplicates. Relative gene expression levels were computed by the 2−∆∆CT
method, where ∆∆CT = ∆CT (mutant) − ∆CT (WT), ∆CT = CT (target gene) − CT (gyrA),
and CT is the threshold cycle value of the amplified gene.

2.5. Bioinformatics Analysis

Computational analyses, including gene ontology and KEGG pathway enrichment
analysis, were performed [26]. Pooled RNA-seq data were used to compare genome-wide
SNP and InDel results between the mutant 1801_2010 and WT Newman_2010 strains. The
details are referenced [27].

2.6. Data Accession Numbers

Project accessions in NCBI: PRJNA235858 and PRJNA235865 for S. aureus newman_2010
and 1801_2010 strains are available, respectively. DNA sequence accession numbers are
SRX478042 (Newman_2010) and SRX478056 (1801_2010). The whole-genome sequence of S.
aureus Newman_WT (GenBank accession: NC_009641) was used as a reference for genome
mapping.

3. Results
3.1. Integrated Genome and Transcriptome Sequencing for Identification of Genetic Variants
In this study, we applied a Circos diagram (Figure 1) to illuminate the whole-genome
and transcriptome sequencing in order to graphically represent genetic variations and
association with their corresponding phenotypes across S. aureus Newman WT and mutant
strains. Figure 1 shows several genetic differences between mutant 1801_2010 and WT
Newman_2010 compared to the reference genome of Newman_WT (NC_009641).
Antibiotics 2022, 11, x FOR PEER REVIEW 4 of 14

association with their corresponding phenotypes across S. aureus Newman WT and mu-
Antibiotics 2022, 11, 125 tant strains. Figure 1 shows several genetic differences between mutant 1801_2010 and4 of 13
WT Newman_2010 compared to the reference genome of Newman_WT (NC_009641).

Figure 1. WGS and RNA-seq polymorphism comparison. Ring 1 (outer circle) shows the chromo-
somal map positions; ring 2 lists chromosome genes; rings 3 and 4 represent the SNP differences
between S. aureus mutant 1801_2010 to Newman_WT and Newman_2010 to Newman_WT; rings
5 and 6 represent the InDel differences between S. aureus mutant 1801_2010 to Newman_WT and
Figure 1. WGS and RNA-seq
Newman_2010 polymorphism comparison. Ring 1 (outer circle) shows the chromoso-
to Newman_WT.
mal map positions; ring 2 lists chromosome genes; rings 3 and 4 represent the SNP differences be-
3.2.
tween S. InDels, Gaps, and
aureus mutant SNPs Identified
1801_2010 to Newman_WT and Newman_2010 to Newman_WT; rings 5
and 6 represent the InDel differences
There was a total of 397 SNPs,between
1925S.gaps,
aureus mutant
and 1801_2010
26 InDels to Newman_WT
detected and
in mutant 1801_2010
Newman_2010 to Newman_WT.
and 360 SNPs, 2185 gaps, and 21 InDels in WT Newman_2010 (Figure 2) compared to
Newman_WT via using the EDGE bioinformatics software [27]. RNA-seq data were used
3.2. InDels, Gaps,
to verify andresults.
these SNPs Identified
The purpose of this study is to investigate whether these genomic
There was contribute
alterations a total of 397 SNPs,
to gene 1925 gaps,There
expression. and 26 InDels
are detected
six rings in mutant
in Figure 1801_2010
1: the inner two rings
and 360
are SNPs, 2185 gaps,
the genetic and 21
difference ofInDels
InDels,inthe
WTmiddle
Newman_2010
two rings(Figure
are SNP 2)differences.
compared toThe New-
second
man_WT via using
ring from the EDGE
the outside is thebioinformatics
list of all genes.software [27].variations
All genetic RNA-seqofdata SNPs were
andused
InDelstofrom
WT
verify Newman_2010
these results. The and mutantof1801_2010
purpose this studywere
is tomapped
investigaterelative to thethese
whether reference genome
genomic
sequence
alterations of Newman_WT
contribute (NC_009641).
to gene expression. ThereSeveral
are siximportant genes have
rings in Figure 1: thebeen
inneridentified
two
ringswhich
are thecould be responsible
genetic difference of forInDels,
the phenotype.
the middleTable 1 listsare
two rings theSNP
polymorphic
differences.changes
The in
the ring
second mutantfromcompared
the outsideto is
Newman_WT.
the list of all Of the 21
genes. Allsignificant SNPs, 18ofwere
genetic variations SNPsassociated
and
with
InDels from4 individual genes (NWMN_0305,
WT Newman_2010 NWMN_0306,
and mutant 1801_2010 wereNWMN_0979,
mapped relative andtoNWMN_1288).
the refer-
The remaining three SNPs were distributed in the intergenic
ence genome sequence of Newman_WT (NC_009641). Several important genes have regions: two of them
beenwere
between the lctp and Spa genes and one was located between
identified which could be responsible for the phenotype. Table 1 lists the polymorphic the recR and tmk genes.
Table 2 presents the 15 InDels identified in mutant 1801_2010
changes in the mutant compared to Newman_WT. Of the 21 significant SNPs, 18 were compared to Newman_WT.
Two genes
associated with(NWMN_1308
4 individual and genes NWMN_1410)
(NWMN_0305, contained 5 InDels. The
NWMN_0306, remaining 10
NWMN_0979, InDels
and
were distributed in 5 important intergenic regions: 4 of them were in the intergenic region
between the NWMN_0810 and NWMN_0811 genes; 2 were in the intergenic region between
the NWMN_2500 and ldh genes; 2 of the InDels were in the intergenic region between the
citZ and aapA genes; 2 of them were in the region between dnaA and rpmH. In Tables 1
 the intergenic region between the NWMN_0810 and NWMN_0811 gen
intergenic region between the NWMN_2500 and ldh genes; 2 of the In
intergenic region between the citZ and aapA genes; 2 of them were in the
dnaA and rpmH. In Tables 1 and 2, two different assembly methods w
Antibiotics 2022, 11, 125 identify genetic variations: one was based on sequence reads5 and
of 13 the oth

contigs. The results from both methods agreed with each other very well

and 2, two different assembly methods were compared to identify genetic variations: one
was based on sequence reads and the other was based on contigs. The results from both
methods agreed with each other very well.

Figure 2. Summary statistics for the InDel and SNP data of S. aureus Newman_2010 and mutant
1801_2010 by comparing to Newman_WT (NC_009641).

Figure
3.3. Identification 2. Summary
of Up-Regulated andstatistics for theGenes
Down-Regulated InDel and SNP data of S. aureus Newman
1801_2010 by comparing to Newman_WT
After normalization, the DESeq2 tool [28] was employed (NC_009641).
for quantitative analysis of
RNA-seq data and identification of the genes differentially expressed between the mutant
and WT strains. Table 1. Polymorphic
In total, changesexpressed
1091 differentially in S. aureus mutant
genes 1801_2010
(DEGs) (padj < compared
0.001) wereto Newm
obtained (Supplementary Table S1): 18 of them were significantly up-regulated (padj < 0.001,
Technol- SNP_po- Synony-
Sample log2 FC > 0.7,
Method FDR < 0.05) and 6 genesSub_codon
Ref_codon were significantly
Ref_aadown-regulated
Sub_aa (padj < 0.001, log2
Product CD
ogy sition
FC < −0.7, FDR < 0.05). The summary of DEGs between the mutant mous
1801_2010 and WT
Newman_2010 is shown in Table 3. Comparative transcriptome analysis and de novo Intergenic
short-readregion
WGS mutant transcriptome
contigs assembly revealed
73,365 C that significant
T transcriptional changes in response
(betweento the
lctP and
triple-acting PGH 1801 were associated with membrane, cell wall, and their related genes spa)
(e.g., amidase, peptidase, holin, and phospholipase D/transphosphatidylase).
Antibiotics 2022, 11, 125 6 of 13

Table 1. Polymorphic changes in S. aureus mutant 1801_2010 compared to Newman_WT *.

Technology Sample Method SNP_position Ref_codon Sub_codon Ref_aa Sub_aa Synonymous Product CDS_start CDS_end
WGS mutant contigs 73,365 C T Intergenic region (between lctP and spa)
WGS mutant reads 73,365 C T Intergenic region (between lctP and spa)
WGS mutant contigs 354,546 GCG GCA A A Yes NWMN_0305: hypothetical protein 353,131 355,029
WGS mutant contigs 354,585 GGT GGG G G Yes NWMN_0305: hypothetical protein 353,131 355,029
WGS mutant contigs 354,594 TTT TTG F L No NWMN_0305: hypothetical protein 353,131 355,029
WGS mutant contigs 354,729 TGT TGC C C Yes NWMN_0305: hypothetical protein 353,131 355,029
WGS mutant contigs 354,767 AAT AGT N S No NWMN_0305: hypothetical protein 353,131 355,029
WGS mutant contigs 354,859 TTT CTT F L No NWMN_0305: hypothetical protein 353,131 355,029
WGS mutant contigs 354,906 CCG CCA P P Yes NWMN_0305: hypothetical protein 353,131 355,029
WGS mutant contigs 355,079 ACA ACG T T Yes NWMN_0306: hypothetical protein 355,029 356,852
WGS mutant contigs 355,208 AAG AAA K K Yes NWMN_0306: hypothetical protein 355,029 356,852
WGS mutant contigs 355,674 GTC TTC V F No NWMN_0306: hypothetical protein 355,029 356,852
WGS mutant contigs 504,664 T C Intergenic region (between recR and tmk)
WGS mutant contigs 1,086,625 AGC AGT S S Yes NWMN_0979 (pycA): pyruvate carboxylase 1,085,501 1,088,971
WGS mutant reads 1,086,625 AGC AGT S S Yes NWMN_0979 (pycA): pyruvate carboxylase 1,085,501 1,088,971
RNA-seq mutant contig 1,086,625 AGC AGT S S Yes NWMN_0979 (pycA): pyruvate carboxylase 1,085,501 1,088,971
RNA-seq mutant reads 1,086,625 AGC AGT S S Yes NWMN_0979 (pycA): pyruvate carboxylase 1,085,501 1,088,971
WGS mutant contigs 1,420,272 ATT GTT I V No NWMN_1288: hypothetical protein 1,420,254 1,421,021
WGS mutant reads 1,420,272 ATT GTT I V No NWMN_1288: hypothetical protein 1,420,254 1,421,021
RNA-seq mutant contig 1,420,272 ATT GTT I V No NWMN_1288: hypothetical protein 1,420,254 1,421,021
RNA-seq mutant reads 1,420,272 ATT GTT I V No NWMN_1288: hypothetical protein 1,420,254 1,421,021
* A wild-type Newman strain (NC_009641) was used as a reference in the data analysis.
Antibiotics 2022, 11, 125 7 of 13

Table 2. InDels identified in S. aureus mutant 1801_2010 compared to Newman_WT *.

Technology Sample Method InDel_position Sequence InDel_seq Length Type Product CDS_start CDS_end
Intergenic region (between dnaA
DNAseq WT reads 2 GAT GATCGAT 4 Insertion
and rpmH)
Intergenic region (between dnaA
DNAseq mutant reads 4 TT TTTTTATCGATT 10 Insertion
and rpmH)
Intergenic region (between
RNA-seq mutant reads 897,844 GC GCC 1 Insertion
NWMN_0810 and NWMN_0811)
Intergenic region (between
DNAseq mutant reads 897,844 GC GCC 1 Insertion
NWMN_0810 and NWMN_0811)
Intergenic region (between
DNAseq mutant reads 897,951 TG TGG 1 Insertion
NWMN_0810 and NWMN_0811)
Intergenic region (between
RNA-seq mutant reads 897,989 AT ATT 1 Insertion
NWMN_0810 and NWMN_0811)
NWMN_1308 (dapD)
DNAseq mutant reads 1,441,311 ACCC ACC 1 Deletion 1,440,676 1,441,395
:tetrahydrodipicolinate acetyltransferase
NWMN_1308 (dapD)
DNAseq mutant reads 1,441,331 CA CAA 1 Insertion 1,440,676 1,441,395
:tetrahydrodipicolinate acetyltransferase
DNAseq mutant reads 1,578,835 GAAA GAA 1 Deletion NWMN_1410:pyrroline-5- carboxylate reductase 1,578,193 1,579,008
RNA-seq mutant reads 1,578,900 GCC GC 1 Deletion NWMN_1410:pyrroline-5- carboxylate reductase 1,578,193 1,579,008
DNAseq mutant reads 1,578,900 GCC GC 1 Deletion NWMN_1410:pyrroline-5- carboxylate reductase 1,578,193 1,579,008
Intergenic region (between citZ and
DNAseq mutant reads 1,761,003 ATTTTTT ATTTTT 1 Deletion
aapA)
Intergenic region (between citZ and
DNAseq mutant contigs 1,761,008 T 1 Deletion
aapA)
Intergenic region (between
RNA-seq mutant contigs 2,744,980 G 1 Deletion
NWMN_2500 and Ldh)
Intergenic region (between
DNAseq mutant contigs 2,744,980 G 1 Deletion
NWMN_2500 and Ldh)
CTTTTA CTTTTATCGATTT Intergenic region (between dnaA
DNAseq mutant reads 2,878,891 9 Insertion
T TAT and rpmH)
* A wild-type Newman strain (NC_009641) was used as a reference in the data analysis.
Antibiotics 2022, 11, 125 8 of 13

Table 3. Up- and down-regulated genes in S. aureus mutant 1801_2010 compared to WT New-
man_2010.

Down-regulated gene (higher expression in WT)

Gene_symbol Gene_ID LogFC LogCPM p-Value FDR Protein name
NWMN_0078 BAF66350 −0.83 7.32 0.00 0.01 surface protein SasD
gntR BAF66470 −0.92 5.12 0.00 0.03 GntR
NWMN_0738 BA67010 −0.71 8.16 0.00 0.04 Conserved hypothetical protein
NWMN_1951 BAF68223 −1.08 4.81 0.00 0.04 oxidoreductase
lukF BAF68199 −0.95 6.15 0.00 0.04 gamma-hemolysin subunit B
NWMN_2209 BAF68481 −0.83 6.91 0.00 0.04 conserved hypothetical protein
Up-regulated gene (lower expression in WT)
NWMN_2304 BAF68576 3.13 4.99 0.00 0.00 membrane protein
holin (holin, toxin
NWMN_1882 BAF68154 1.85 4.17 0.00 0.00 secretion/phage lysis family
protein)
NWMN_0537 BAF66809 1.05 11.68 0.00 0.00 membrane protein
NWMN_1068 BAF67340 1.21 4.88 0.00 0.00 conserved hypothetical protein
NWMN_0909 BAF67181 2.88 2.60 0.00 0.00 membrane protein
NWMN_2505 BAF68777 1.69 4.27 0.00 0.00 membrane protein
NWMN_1874 BAF68146 1.18 5.74 0.00 0.01 putative membrane protein
NWMN_2287 BAF68559 0.69 7.66 0.00 0.01 hsp20-like protein
NWMN_1639 BAF67911 0.77 6.91 0.00 0.01 peptidase
cytochrome C biogenesis protein
NWMN_1256 BAF67528 0.72 6.40 0.00 0.03
CcdC
NWMN_0985 BAF67257 0.91 5.07 0.00 0.03 conserved hypothetical protein
nrdH BAF67223 1.24 4.55 0.00 0.04 NrdH-redoxin
NWMN_2223 BAF68495 0.73 6.62 0.00 0.04 conserved hypothetical protein
NWMN_1881 BAF68153 0.78 6.25 0.00 0.04 amidase
NWMN_2154 BAF68426 0.83 6.67 0.00 0.04 probable membrane protein
NWMN_0920 BAF67192 0.99 4.49 0.00 0.04 acyltransferase
NWMN_0986 BAF67258 0.81 5.86 0.00 0.05 conserved hypothetical protein
phospholipase
NWMN_1229 BAF67501 1.43 3.40 0.00 0.05
D/transphosphatidylase

3.4. Function Ontology and KEGG Pathway Enrichment Analyses of DEGs

To annotate the potential functions of the DEGs between WT and mutant strains,
DEGs with >2-fold expression change were assigned to different KEGG pathways. All
KEGG pathways were analyzed as shown in Figure 3. In these pathways, energy pro-
duction and conversion, amino acid transport and metabolism, nucleotide transport and
metabolism, intracellular trafficking, secretion, and vesicular transport, signal transduction
and mechanisms were the most enriched pathways either up- or down-expressed between
WT Newman_2010 and mutant 1801_2010 strains.

3.5. RT-qPCR Confirmation

To verify the most differentially expressed genes obtained from RNA-seq datasets
of WT Newman_2010 and mutant 1801_2010, we performed reverse transcription qPCR
experiments and compared these results with RNA-seq. Figure 4 demonstrated that the
changes of gene expression from RT-qPCR correlated well with the transcriptome profiling
from RNA-seq. Moreover, these results were consistent with the observed structural
changes at the DNA and RNA levels (Tables 1 and 2). These structural changes in the
 NWMN_0986 BAF67258 0.81 5.86 0.00 0.05 conserved hypothetical protein
NWMN_1229 BAF67501 1.43 3.40 0.00 0.05 phospholipase D/transphosphatidylase

3.4. Function Ontology and KEGG Pathway Enrichment Analyses of DEGs

To annotate the potential functions of the DEGs between WT and mutant strains,
Antibiotics 2022, 11, 125 DEGs with >2-fold expression change were assigned to different KEGG pathways. All 9 of 13
KEGG pathways were analyzed as shown in Figure 3. In these pathways, energy produc-
tion and conversion, amino acid transport and metabolism, nucleotide transport and me-
tabolism, intracellular trafficking, secretion, and vesicular transport, signal transduction
genes encoding membrane/cell surface proteins and the perturbation in gene expression
and mechanisms were the most enriched pathways either up- or down-expressed between
are potential candidates responsible for resistance to these novel antimicrobials.
WT Newman_2010 and mutant 1801_2010 strains.

Antibiotics 2022, 11, x FOR PEER REVIEW 10 of 14

changes of gene expression from RT-qPCR correlated well with the transcriptome profil-
ingFigure
from3.RNA-seq. Moreover, these results were consistent with the observed structural
KEGG pathway classification of the DEGs between S. aureus mutant 1801_2010 and WT
Figure
changes 3. KEGG
at the
Newman_2010. pathway
DNA andclassification
RNA levels of the
(Tables and 2). S.These
DEGs1between aureusstructural
mutant 1801_2010
changesand
in WT
the
Newman_2010.
genes encoding membrane/cell surface proteins and the perturbation in gene expression
are3.5. RT-qPCR
potential Confirmation
candidates responsible for resistance to these novel antimicrobials.
To verify the most differentially expressed genes obtained from RNA-seq datasets of
WT Newman_2010 and mutant 1801_2010, we performed reverse transcription qPCR ex-
periments and compared these results with RNA-seq. Figure 4 demonstrated that the

Figure 4. Cont.
Antibiotics 2022, 11, 125 10 of 13
Antibiotics 2022, 11, x FOR PEER REVIEW 11 of 14

Figure 4. Validation of RNA-seq analysis by RT-qPCR. Relative gene expression levels of S. aureus
Validation
Figure 4.1801_2010
mutant of RNA-seq
and analysis bywere
WT Newman_2010 RT-qPCR. Relative
quantified gene expression
by RT-qPCR, and datalevels S. aureus
ofanalyzed
were
mutant
using 1801_2010
the and critical
comparative WT Newman_2010 were method.
threshold (ΔΔCT) quantifiedThebyratios
RT-qPCR,
(log2)and data were
of relative geneanalyzed
expres-
usingfrom
sion the comparative
RT-qPCR and critical threshold
RNA-seq (∆∆CT)inmethod.
are shown grey and The ratios
black (log2)
bars, of relative gene
respectively. expression
A ratio greater
than
from zero (>0) indicates
RT-qPCR up regulation
and RNA-seq are shownofingene
greyexpression
and black and
bars,a respectively.
ratio below zero (<0)greater
A ratio indicates
thandown
zero
regulation in mutant
(>0) indicates 1801_2010.
up regulation Error
of gene bars indicate
expression and astandard
ratio belowdeviations
zero (<0)ofindicates
three replicates.
down regulation
in mutant 1801_2010. Error bars indicate standard deviations of three replicates.
4. Discussion
4. Discussion
Our previous genome sequencing work
Our previous genome sequencing work (https://www.ncbi.nlm.nih.gov/sra/?term=
(https://www.ncbi.nlm.nih.gov/sra/?term=SRX478056) (accessed on 12 January 2022) has
SRX478056)
been trying to(accessed
identifyonthe10genomic
Januarychanges
2022) hasinbeen trying to identify
Staphylococcus the genomic
aureus that changes
confer resistance
in Staphylococcus aureus that confer resistance to peptidoglycan hydrolase
to peptidoglycan hydrolase antimicrobial enzymes [15,29]. In this study, we combined antimicrobial en-
zymes [15,29]. In this study, we combined RNA-seq and genome
RNA-seq and genome sequence data of WT Newman_2010 and mutant 1801_2010 strains sequence data of WT
Newman_2010
by and mutant 1801_2010
comparing genome-wide strains
SNP and InDel by comparing
results. genome-wide
Additionally, we appliedSNP and InDel
RT-qPCR to
results. Additionally, we applied RT-qPCR to confirm these results. To reveal
confirm these results. To reveal the differences between the positive genes and other the differences
between
genes, the positive genes
computational and other
analyses, genes,gene
including computational analyses,
ontology and KEGG including
pathwaygene ontology
enrichment,
and KEGG
were pathway enrichment,
also employed. were also
These differences employed.
between mutant These differences
1801_2010, WT between mutant
Newman_2010,
1801_2010, WT Newman_2010, and Newman_WT strains in Figure
and Newman_WT strains in Figure 1 indicate these genetic changes are potentially1 indicate these genetic
re-
changes are potentially responsible for phenotypic variation. However, a phenotypic
sponsible for phenotypic variation. However, a phenotypic trait caused by genetic sources trait
caused by genetic sources of variation could include additive variance, dominant variance,
of variation could include additive variance, dominant variance, environmental variance
environmental variance (e.g., organismal adaptation), and epistatic variance [30,31].
(e.g., organismal adaptation), and epistatic variance [30,31].
In Tables 1 and 2, and Figure 2, we observed the genetic variations which could be
In Tables 1 and 2, and Figure 2, we observed the genetic variations which could be
responsible for the mechanism of resistance to peptidoglycan hydrolase (PGH 1801) in
responsible for the mechanism of resistance to peptidoglycan hydrolase (PGH 1801) in S.
S. aureus. However, these variations are only statistical indicators of a functional effect
aureus. However, these variations are only statistical indicators of a functional effect asso-
ciated with their genotypic variants because it is uncommon to have a concrete variant
Antibiotics 2022, 11, 125 11 of 13

associated with their genotypic variants because it is uncommon to have a concrete variant
with a “precise” genetic location with measurable statistical “effects”. Although we can say
that there is a functional effect, in order to determine functionality we must go beyond the
identification of the variant phenotype due to a specific SNP- or InDel-based locus. It will
be instructive to compare the results of current study with those in the literatures. Mostly,
there are genomic loci that could influence the expression level of mRNA and these loci
can be physically located close to or far away from the gene that gets regulated. It is not
necessary that genetic loci are associated with a SNP or InDel.
In this study, we observed significant transcriptional changes in S. aureus New-
man_2010 upon exposure to the triple-acting fusion protein PGH1801 [2,12]. We found most
of these are membrane proteins, cell wall related proteins [32–37], such as amidase [12],
peptidase [29], holin [38–42], and phospholipase D/transphosphatidylase [43,44].
Through the Gene Ontology function and KEGG pathway enrichment analyses, we
found energy production and conversion, amino acid transport and metabolism, nucleotide
transport and metabolism, intracellular trafficking, secretion, and vesicular transport,
signal transduction and mechanisms were the most enriched pathways either up- or down-
expressed between the wild-type and mutant.
Comparative transcriptome analysis and de novo short-read assembly in this study
revealed that the genes with significant transcriptional changes in response to exposure to
the triple-acting fusion protein are associated with membrane and cell wall (e.g., amidase,
peptidase, holin, and phospholipase D/transphosphatidylase). These results are consistent
with the observed nucleotide changes at the DNA level. The nucleotide changes in the
genes encoding membrane/cell surface proteins and the alteration of gene expression may
contribute to the increased resistance of S. aureus to PGHs. The findings of this study could
provide insights into the target genes responsible for PGH resistance and lead to the design
of new antimicrobial agents.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/antibiotics11020125/s1, Figure S1: Growth curves of S.
aureus Newman_2010 and mutant 1801_2010; Table S1: Complete list of differentially expressed genes
based on normalized RNA-seq data.
Author Contributions: X.Y. and Y.H.—planned and directed experiments, analyzed results, and
prepared and edited the manuscript; Y.X.—designed and conducted experiments, processed data,
and assisted in manuscript preparation, C.L., D.M.D., A.G. and P.I.—planned experiments, analyzed
data, and edited the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was supported by the U.S. Department of Agriculture, Agricultural Research
Service (USDA-ARS), Current Research Information System number 8072-42000-093 8072-42000-093.
Data Availability Statement: DNA sequence reads are available in GenBank of NCBI with the
accession No. SRX478042 for S. aureus Newman_2010 and SRX478056 for mutant 1801_2010. The
GenBank accession number for complete genome sequence of S. aureus Newman_WT is NC_009641.
Conflicts of Interest: The authors declare that they have no conflict of interest.

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