# Impact of Deficiencies in Branched-Chain Fatty Acids and Staphyloxanthin inStaphylococcus aureus

Authors: Hannah Braungardt; Vineet K. Singh
Journal: BioMed Research International
 (2019)

---

## Abstract

Staphylococcus aureusis a well-known human pathogen with the ability to cause mild superficial skin infections to serious deep-tissue infections, such as osteomyelitis, pneumonia, and infective endocarditis. A key toS. aureusinfections and its pathogenicity is its ability to survive in adverse environments, especially at lower temperatures, by regulation of its cell membrane. Branched-chain fatty acids (BCFAs) and staphyloxanthin have been shown to regulate membrane fluidity and staphylococcal virulence. This study was conducted with the hypothesis that the simultaneous lack of BCFAs and staphyloxanthin will have a far greater implication on environmental survival and virulence ofS. aureus. Lack of a functional branched-chain α-keto acid dehydrogenase (BKD) enzyme because of a mutation in thelpdA gene led to a decrease in the production of BCFAs, membrane fluidity, slower growth, and poorin vivo survival ofS. aureus. A mutation in thecrtMgene eliminated the production of staphyloxanthin but it did not affect membrane BCFA levels, fluidity, growth, orin vivo survival. AcrtM:lpdAdouble mutant showed much slower growth and attenuation compared to individual mutants. The results of this study suggest that simultaneous targeting of the BCFA and staphyloxanthin biosynthetic pathways can be a strategy to controlS. aureusinfections.


---

## Body

## 1. Introduction

S. aureusis a common cause of skin infections and is also responsible for a myriad of complicated deep-tissue diseases such as pneumonia, endocarditis, osteomyelitis, and septic shock [1–3]. Treatment ofS. aureusinfections can be difficult due to its antibiotic resistance. Infections caused by methicillin-resistantS. aureus(MRSA) and vancomycin resistantS. aureus(VRSA) have limited treatment options [4–6]. As an alternative strategy, metabolic pathways critical for survival and virulence can be targeted as ways to reduceS. aureusinfections and their severity [6].In order to survive in harsh environments, bacterial pathogens may alter the fatty acid composition of their membrane to maintain homeostasis [7–9]. Fatty acids inS. aureuscell membranes include saturated- and unsaturated-straight chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) [10–12]. Levels of these fatty acids, especially BCFAs, change in response to fluctuations in growth environments such as temperature, humidity, and nutrient availability [10]. The enzyme, branched-chain α-keto acid dehydrogenase (BKD), catalyzes the committed step in the metabolic pathway that yields BCFAs. Lack of BKD enzymatic activity decreases BCFAs inS. aureuscell membrane which hinders the growth of this bacterium at low temperatures [11, 12].An additional key regulator ofS. aureusvirulence and membrane fluidity is the carotenoid pigment, staphyloxanthin [13, 14]. Its production is mediated by enzymes encoded by five genes (crtOPQMN) present in a cluster in the staphylococcal chromosome [13, 14]. Inactivation of the genes in this cluster abolishes staphyloxanthin production which results in colorless colonies of this bacterium [13].While a higher level of BCFAs leads to an increase in fluidity, an increase in carotenoid production decreases the membrane fluidity [15–18].S. aureuscells deficient in staphyloxanthin are more susceptible to the killing by neutrophils and are more easily cleared from infected tissues thanS. aureuswith staphyloxanthin [13]. It has been postulated that staphyloxanthin plays an important role inS. aureus virulence by conferring protection of the bacterial cells from oxidants generated during respiratory burst inside the phagocytic cells [13]. Recently, the staphyloxanthin pathway has been studied for its potential as a drug target to controlS. aureus infections [19].We recently noted an increased production of staphyloxanthin in BKD-deficientS. aureus cells with decreased level of BCFAs [12]. It was speculated that this increase in staphyloxanthin production compensated for a more rigid membrane due to a decreased membrane BCFAs [12]. The findings of this study highlight the roles of BCFAs and staphyloxanthin and their significance as antimicrobial targets to control infections caused byS. aureus.


## 2. Materials and Methods

### 2.1. Ethics Statement

Animal studies were conducted as per a protocol approved by the Institutional Animal Care and Use Committee of A.T. Still University-Kirksville College of Osteopathic Medicine.


### 2.2. Bacterial Strains, Antibiotics, and Growth Conditions

The bacterial strains used in this study are shown in Table1. All cultures were grown aerobically at 37°C in tryptic soy broth (TSB) or on tryptic soy agar (TSA). Antibiotics, erythromycin (10 μg ml−1) and kanamycin (100 μg ml−1), were used when needed. Additionally, liquid bacterial cultures were grown in a shaking incubator at 220 rpm, and plated cultures were incubated for 24-48 h.Table 1
Bacterial strains used in this study.
Strains Characteristics Reference SH1000 A methicillin-sensitive and SigB positiveS. aureus [20]  SH1000:lpdA lpdAmutant of S. aureus strain SH1000 (KanR) [11]  SH1000:crtM Mutation incrtM gene transduced from NE1444 to S. aureus SH1000 (ErmR) This study  SH1000:crtM:lpdA AcrtM:lpdA double mutant of S. aureus SH1000 (KanR, ErmR) This study  SH1000:lpdA+pCU-BKD lpdA mutant of SH1000 complemented with BKD gene cluster in trans(KanR, CamR) [11]  SH1000:crtM+pCU-crtOPQMN crtM mutant of SH1000 complemented with crt gene cluster in trans(ErmR, CamR) This study  SH1000:crtM:lpdA+pCU-BKD AcrtM:lpdA double mutant of SH1000 complemented with BKD gene cluster (KanR, ErmR, CamR) This study  SH1000:crtM:lpdA+pCU-crtOPQMN AcrtM:lpdA double mutant of SH1000 complemented with crt gene cluster (KanR, ErmR, CamR) This study


### 2.3. S. aureus crtM and lpdA Mutants

ThelpdAmutant used in this study has previously been described [11]. AcrtMmutant ofS. aureus strain JE2 (NE1444) was obtained from the Nebraska Transposon Mutant Library. Mutation incrtM gene was subsequently transferred intoS. aureus strain SH1000 using a phage transduction procedure and verified by PCR. To createcrtM:lpdA double mutant, thecrtM mutation was transduced into thelpdA mutant ofS. aureus strain SH1000.


### 2.4. Complementation oflpdAandcrtM Mutant

Complementation of thelpdAmutant was accomplished as previously described [11]. For complementation of thecrtM mutant, a 6310 bp DNA fragment was PCR amplified using primer pairs (5′-GGTACCATCGCCATTCACCGTTATGT-3′ and 5′-TCTAGATGTTGGTGGGAAAATTGGTT-3′) andS. aureus SH1000 genomic DNA as template. This amplicon represents a fragment starting 436 nt upstream of thecrtO gene, the entire carotenoid pathway encoding genes (crtOPQMN), and 337 nt downstream of thecrtN termination codon. The amplicon was cloned into the shuttle plasmid pCU1 [21] which was then placed into thecrtMmutant strains ofS. aureusstrain SH1000. Complementation was verified by dark pigmentation of thecrtMmutants.


### 2.5. Growth Kinetics of thecrtM,lpdA, andcrtM:lpdAMutants and Wild-Type Strain SH1000

Overnight cultures of these strains were diluted 1:10 and the absorbance of the diluted culture was measured spectrophotometrically. These overnight cultures were then diluted in a 250 ml Erlenmeyer flasks containing 50 ml of fresh TSB with loose metal caps for proper aeration. Dilutions were made 1:100 if the overnight cultures showed an OD600 of 6.0 or adjusted accordingly. The culture flasks were placed in a shaking incubator. Optical density (OD600) of each culture was determined spectrophotometrically every 30 minutes and continued until cultures reached the stationary phase. The generation time of growth of differentS. aureus strains was calculated as previously described [22]. The growth rate (μ) was calculated using the formula (ln OD2 - ln OD1)/(t2 - t1) in which two time point ODs from 120 (t1) and 210 min (t2) were used, and the generation time was then calculated using the formula (ln 2/μ) as described previously [22].


### 2.6. Analysis of the Membrane Fatty Acid Composition incrtM,lpdA, andcrtM:lpdA Mutants and the Parent SH1000

The membrane fatty acid composition was determined as previously described [11, 12]. Bacterial cells grown in TSB were harvested during mid-exponential growth phase (OD600 = 0.6). After washing three times with cold distilled water, cell pellets were frozen and shipped under dry ice for the analysis of membrane fatty acids to Midi Microbial Identification system (Newark, DE).


### 2.7. Carotenoid Levels incrtM:lpdADouble Mutant and the Complemented Strains

Quantification of the carotenoid pigment was carried out using a methanol extraction procedure as previously described [10]. Overnight cultures were diluted (1:100) and added to a flask containing 50.0 ml fresh TSB. The cultures were allowed to grow shaking at 37°C for 48 h. Cells from 5.0 ml culture were pelleted by centrifugation and washed twice with cold water, and the wet mass of the bacterial cell pellet was measured. Carotenoid pigments from these cells were then extracted after resuspension in 3.0 ml of methanol and incubation at 55°C for 3 min with intermittent agitation. A465 of the cell-free supernatant was then measured as level of carotenoid production, which was then normalized against the wet bacterial cell mass.


### 2.8. Determination of Membrane Fluidity of thecrtM,lpdA, andcrtM:lpdAMutants and Wild-Type Strain SH1000

Membrane fluidity was measured as recently described [12]. Overnight cultures were diluted 1:100 and were grown at 37°C with shaking until an OD600 of 1.0 was reached. The cultures were then centrifuged to collect the bacterial cells that were washed twice with phosphate buffered saline (PBS) solution before being resuspended in PBS to an OD600 of 1.0. To this suspension, 4 μl of 1,6-diphenyl-1,3,5-hexatriene (DPH) was added and the fluorescence polarization was measured using a spectrofluorometer (Fluorescence Spectrometer LS-55; Llantrisant, UK).


### 2.9. Survival oflpdA,crtM, andcrtM:lpdAMutants in Comparison to Wild-Type SH1000 in Mice

In vivo survival experiments were carried out as recently described [23]. Cultures of these strains were grown individually to mid-log phase (OD600 = 0.6) in TSB. Bacterial cells were then harvested by centrifugation, washed three times with 1% TSB, and resuspended in 1% TSB. Cell viability in the suspension was determined by serial dilution and plating. Three parallel mixtures of wild-type withlpdA,crtM, orcrtM:lpdA mutants were prepared (1X107 CFU ml−1; ~60/40 percent mixture of mutant/wild-type) in 1% TSB, and 0.2 ml of this suspension was injected into the peritoneal cavity of Swiss white Hla (ICR) CVF female mice (16-20 gram) (Hilltop Lab Animals, Inc.) with a 26-gauge needle fitted to a 1 ml syringe. At 8 and 24 h, the mice were euthanized and the liver and spleen were aseptically removed. Excised livers and spleens were homogenized in 2 and 1 ml of 1% TSB, respectively, using a glass tissue grinder fitted with a glass pestle. The tissue homogenates were serially diluted, plated on TSA and TSA containing appropriate antibiotics, and allowed to grow overnight by incubation at 37°C. The bacterial colonies growing in the presence of antibiotics were compared to the total number of colonies on TSA plates to calculate the fraction of mutants in the bacterial population in the infected tissues. The ratio of mutants in the infected tissues was then compared to the ratio of mutants in the inoculum used to infect the mice as previously described [23].


### 2.10. Statistical Analysis

Significance is determined using a one-way ANOVA and with ap value ≤ 0.05. For wild-type versus mutant in the mouse survival experiment, data were evaluated using one-sided proportion ratio with a Bonferroni correction using R studio.


## 2.1. Ethics Statement

Animal studies were conducted as per a protocol approved by the Institutional Animal Care and Use Committee of A.T. Still University-Kirksville College of Osteopathic Medicine.


## 2.2. Bacterial Strains, Antibiotics, and Growth Conditions

The bacterial strains used in this study are shown in Table1. All cultures were grown aerobically at 37°C in tryptic soy broth (TSB) or on tryptic soy agar (TSA). Antibiotics, erythromycin (10 μg ml−1) and kanamycin (100 μg ml−1), were used when needed. Additionally, liquid bacterial cultures were grown in a shaking incubator at 220 rpm, and plated cultures were incubated for 24-48 h.Table 1
Bacterial strains used in this study.
Strains Characteristics Reference SH1000 A methicillin-sensitive and SigB positiveS. aureus [20]  SH1000:lpdA lpdAmutant of S. aureus strain SH1000 (KanR) [11]  SH1000:crtM Mutation incrtM gene transduced from NE1444 to S. aureus SH1000 (ErmR) This study  SH1000:crtM:lpdA AcrtM:lpdA double mutant of S. aureus SH1000 (KanR, ErmR) This study  SH1000:lpdA+pCU-BKD lpdA mutant of SH1000 complemented with BKD gene cluster in trans(KanR, CamR) [11]  SH1000:crtM+pCU-crtOPQMN crtM mutant of SH1000 complemented with crt gene cluster in trans(ErmR, CamR) This study  SH1000:crtM:lpdA+pCU-BKD AcrtM:lpdA double mutant of SH1000 complemented with BKD gene cluster (KanR, ErmR, CamR) This study  SH1000:crtM:lpdA+pCU-crtOPQMN AcrtM:lpdA double mutant of SH1000 complemented with crt gene cluster (KanR, ErmR, CamR) This study


## 2.3. S. aureus crtM and lpdA Mutants

ThelpdAmutant used in this study has previously been described [11]. AcrtMmutant ofS. aureus strain JE2 (NE1444) was obtained from the Nebraska Transposon Mutant Library. Mutation incrtM gene was subsequently transferred intoS. aureus strain SH1000 using a phage transduction procedure and verified by PCR. To createcrtM:lpdA double mutant, thecrtM mutation was transduced into thelpdA mutant ofS. aureus strain SH1000.


## 2.4. Complementation oflpdAandcrtM Mutant

Complementation of thelpdAmutant was accomplished as previously described [11]. For complementation of thecrtM mutant, a 6310 bp DNA fragment was PCR amplified using primer pairs (5′-GGTACCATCGCCATTCACCGTTATGT-3′ and 5′-TCTAGATGTTGGTGGGAAAATTGGTT-3′) andS. aureus SH1000 genomic DNA as template. This amplicon represents a fragment starting 436 nt upstream of thecrtO gene, the entire carotenoid pathway encoding genes (crtOPQMN), and 337 nt downstream of thecrtN termination codon. The amplicon was cloned into the shuttle plasmid pCU1 [21] which was then placed into thecrtMmutant strains ofS. aureusstrain SH1000. Complementation was verified by dark pigmentation of thecrtMmutants.


## 2.5. Growth Kinetics of thecrtM,lpdA, andcrtM:lpdAMutants and Wild-Type Strain SH1000

Overnight cultures of these strains were diluted 1:10 and the absorbance of the diluted culture was measured spectrophotometrically. These overnight cultures were then diluted in a 250 ml Erlenmeyer flasks containing 50 ml of fresh TSB with loose metal caps for proper aeration. Dilutions were made 1:100 if the overnight cultures showed an OD600 of 6.0 or adjusted accordingly. The culture flasks were placed in a shaking incubator. Optical density (OD600) of each culture was determined spectrophotometrically every 30 minutes and continued until cultures reached the stationary phase. The generation time of growth of differentS. aureus strains was calculated as previously described [22]. The growth rate (μ) was calculated using the formula (ln OD2 - ln OD1)/(t2 - t1) in which two time point ODs from 120 (t1) and 210 min (t2) were used, and the generation time was then calculated using the formula (ln 2/μ) as described previously [22].


## 2.6. Analysis of the Membrane Fatty Acid Composition incrtM,lpdA, andcrtM:lpdA Mutants and the Parent SH1000

The membrane fatty acid composition was determined as previously described [11, 12]. Bacterial cells grown in TSB were harvested during mid-exponential growth phase (OD600 = 0.6). After washing three times with cold distilled water, cell pellets were frozen and shipped under dry ice for the analysis of membrane fatty acids to Midi Microbial Identification system (Newark, DE).


## 2.7. Carotenoid Levels incrtM:lpdADouble Mutant and the Complemented Strains

Quantification of the carotenoid pigment was carried out using a methanol extraction procedure as previously described [10]. Overnight cultures were diluted (1:100) and added to a flask containing 50.0 ml fresh TSB. The cultures were allowed to grow shaking at 37°C for 48 h. Cells from 5.0 ml culture were pelleted by centrifugation and washed twice with cold water, and the wet mass of the bacterial cell pellet was measured. Carotenoid pigments from these cells were then extracted after resuspension in 3.0 ml of methanol and incubation at 55°C for 3 min with intermittent agitation. A465 of the cell-free supernatant was then measured as level of carotenoid production, which was then normalized against the wet bacterial cell mass.


## 2.8. Determination of Membrane Fluidity of thecrtM,lpdA, andcrtM:lpdAMutants and Wild-Type Strain SH1000

Membrane fluidity was measured as recently described [12]. Overnight cultures were diluted 1:100 and were grown at 37°C with shaking until an OD600 of 1.0 was reached. The cultures were then centrifuged to collect the bacterial cells that were washed twice with phosphate buffered saline (PBS) solution before being resuspended in PBS to an OD600 of 1.0. To this suspension, 4 μl of 1,6-diphenyl-1,3,5-hexatriene (DPH) was added and the fluorescence polarization was measured using a spectrofluorometer (Fluorescence Spectrometer LS-55; Llantrisant, UK).


## 2.9. Survival oflpdA,crtM, andcrtM:lpdAMutants in Comparison to Wild-Type SH1000 in Mice

In vivo survival experiments were carried out as recently described [23]. Cultures of these strains were grown individually to mid-log phase (OD600 = 0.6) in TSB. Bacterial cells were then harvested by centrifugation, washed three times with 1% TSB, and resuspended in 1% TSB. Cell viability in the suspension was determined by serial dilution and plating. Three parallel mixtures of wild-type withlpdA,crtM, orcrtM:lpdA mutants were prepared (1X107 CFU ml−1; ~60/40 percent mixture of mutant/wild-type) in 1% TSB, and 0.2 ml of this suspension was injected into the peritoneal cavity of Swiss white Hla (ICR) CVF female mice (16-20 gram) (Hilltop Lab Animals, Inc.) with a 26-gauge needle fitted to a 1 ml syringe. At 8 and 24 h, the mice were euthanized and the liver and spleen were aseptically removed. Excised livers and spleens were homogenized in 2 and 1 ml of 1% TSB, respectively, using a glass tissue grinder fitted with a glass pestle. The tissue homogenates were serially diluted, plated on TSA and TSA containing appropriate antibiotics, and allowed to grow overnight by incubation at 37°C. The bacterial colonies growing in the presence of antibiotics were compared to the total number of colonies on TSA plates to calculate the fraction of mutants in the bacterial population in the infected tissues. The ratio of mutants in the infected tissues was then compared to the ratio of mutants in the inoculum used to infect the mice as previously described [23].


## 2.10. Statistical Analysis

Significance is determined using a one-way ANOVA and with ap value ≤ 0.05. For wild-type versus mutant in the mouse survival experiment, data were evaluated using one-sided proportion ratio with a Bonferroni correction using R studio.


## 3. Results

### 3.1. Growth Kinetics of the Mutant and Wild-Type Strains SH1000

A comparison of the growth kinetics of the wild-type SH1000 and thecrtM andlpdA mutants showed a decreased growth rate of thelpdAmutant (Figure 1, Table 2). On the other hand, thecrtMmutant of theS. aureus strain SH1000 showed no change in growth in comparison to the wild-type (Figure 1, Table 2). The growth of thecrtM:lpdA double mutant was significantly slower than the wild-typeS. aureusas well as thelpdA individual mutant at every time point (Figure 1, Table 2). When thecrtM:lpdAwas complemented with a plasmid containing the entirecrt operon, the growth of the complemented strain remained slower like the growth of thelpdA mutant (Figure 2). However, when thecrtM:lpdAmutant was complemented with the entirebkdgene locus on plasmid pCU1, the growth of the complemented strain was restored to the level of the growth rate of the wild-typeS. aureus strain SH1000 (Figure 2).Table 2
Generation time of differentS. aureus strains.
Strain Generation time (min) SH1000 32.09 SH1000:lpdA 34.49 SH1000:crtM 32.68 SH1000:crtM:lpdA 39.51Figure 1
Growth analysis ofS. aureusstrain SH1000 and derivativelpdA andcrtM mutants.Growth was measured every 30 min (OD600) until stationary phase was reached. Values represent an average of two independent experiments.Figure 2
Growth analysis ofcrtM:lpdA double mutant and the mutant complemented with BKD orcrt gene clusterin trans.Growth was measured every 30 min (OD600) until stationary phase was reached. Values represent an average of two independent experiments.


### 3.2. Analysis of the Membrane Fatty Acid Composition of Wild-TypeS. aureus Strain SH1000 and Its DerivativecrtM,lpdA, andcrtM:lpdAMutants

Cell membrane fatty acid compositions ofS. aureuswild-type SH1000 and its isogenic mutants are shown in Table 3. In wild-typeS. aureus, the BCFAs accounts for 70.7% of the total membrane lipids. Fatty acid profile of thecrtMmutant ofS. aureus SH1000 is similar to the fatty acid profile of the parent strain with about 70% BCFAs (Table 3). ThelpdAmutant, on the other hand, showed a significant deficiency in BCFAs and represented only 35.1% of the membrane lipids (Table 3). Fatty acid profile of thecrtM:lpdAdouble mutant was similar to thelpdA individual mutant ofS. aureus SH1000 with a significantly lower BCFA level (42%) compared to their levels (70.7%) in the wild-typeS. aureus (Table 3).Table 3
Major membrane fatty acids ofS. aureus strains.
Wild-type SH1000 crtMmutant lpdAmutant crtM:lpdA C10:0 0.72 ± 0.20 0.66 ± 0.13 0.64 ± 0.40 0.67 ± 0.39  C12:0 0.60 ± 0.10 0.60 ± 0.09 0.56 ± 0.05 0.55 ± 0.04  C13:0 iso 0.25 ± 0.20 0.23 ± 0.02 0.42 ± 0.17 0.37 ± 0.17  Iso C14:0 2.60 ± 0.40 2.68 ± 0.20 8.13 ± 0.44 10.5 ± 0.33  C14:0 1.55 ± 0.40 1.61 ± 0.21 9.67 ± 1.92 7.62 ± 2.30  Iso C15:0 13.5 ± 0.90 13.59 ± 0.61 3.27 ± 0.05 3.81 ± 0.21  Anteiso C15:0 35.4 ± 2.60 35.5 ± 1.70 17.1 ± 5.93 19.4 ± 5.70  Iso C16:0 3.65 ± 0.30 3.64 ± 0.10 3.28 ± 0.76 4.55 ± 1.30  C16:0 6.20 ± 0.30 6.66 ± 0.22 16.2 ± 2.07 14.9 ± 2.34  Iso C17:0 4.14 ± 0.30 4.00 ± 0.13 0.41 ± 0.06 0.54 ± 0.14  Anteiso C17:0 7.86 ± 0.90 7.28 ± 0.57 1.65 ± 0.86 2.02 ± 1.20  C17:0 0.42 ± 0.03 0.36 ± 0.05 0.66 ± 0.10 0.27 ± 0.08  Iso C18:0 0.89 ± 0.10 0.85 ± 0.02 0.55 ± 0.19 0.66 ± 0.25  C18:0 12.0 ± 0.70 12.6 ± 0.59 20.9 ± 1.58 18.9 ± 2.23  Anteiso C19:0 1.11 ± 0.04 0.96 ± 0.05 0.35 ± 0.04 0.28 ± 0.13  C19:0 0.58 ± 0.02 0.45 ± 0.02 0.75 ± 0.11 1.21 ± 0.19  C20:0 5.44 ± 0.60 5.68 ± 0.89 10.7 ± 1.31 6.47 ± 0.92  Anteiso fatty acids 44.4 43.7 19.0 21.7  Iso fatty acids 26.3 26.2 16.1 20.4  BCFA 70.7 69.9 35.1 42.1


### 3.3. Carotenoid Levels incrtM:lpdADouble Mutant and the Complemented Strains

Inactivation of thecrtMgene resulted in a nonpigmentedS. aureus cell. In these experiments both thecrtMandcrtM:lpdAmutants were white and showed minimal carotenoid production (Figure 3). The wild-typeS. aureus SH1000 and thecrtM:lpdA double mutant complemented withcrt gene locus showed restored levels of staphyloxanthin (Figure 3). Conversely, when thecrtM:lpdA double mutant was complemented with the BKD gene cluster, there was no restoration in the mutant in terms of its ability to produce staphyloxanthin (Figure 3).Figure 3
Staphyloxanthin levels inS. aureusstrain SH1000 and isogenic mutants.Carotenoid pigments were methanol extracted from 48 h grown cultures and estimated spectrophotometrically by quantifying OD465. Values represent average of three independent experiments ± standard deviation (∗ significant atp≤.05).


### 3.4. Membrane Fluidity of thecrtM,lpdA, andcrtM:lpdAMutants and the Wild-TypeS. aureus Strain SH1000

A mutation in thelpdA gene resulted in significant decrease in the membrane fluidity of the mutant bacteria (Figure 4). Polarization value recorded for thelpdA mutant was significantly greater when compared to the polarization value for the wild-typeS. aureus suggesting a more rigid membrane. In contrast, thecrtMmutant showed polarization value slightly lower when compared with the polarization value for the wild-typeS. aureus strain SH1000 cells, but this change was not statistically significant. ThecrtM:lpdAdouble mutant was slightly more fluid than thelpdA mutant, but still significantly less fluid than the wild-typeS. aureus strain SH1000.Figure 4
Membrane fluidity ofS. aureus strain SH1000 and derivativelpdA,crtM, andcrtM:lpdA mutants. Cells from mid-exponential phase cultures were washed and incubated with DPH. Membrane fluidity was measured with a spectrofluorometer. Values represent average of three independent experiments ± standard deviation (∗ significant atp≤.05).


### 3.5. Survival oflpdA,crtM, andcrtM:lpdAMutants in Mice

In these bacterial survival studies in mice, a mixture of wild-type SH1000 with eitherlpdA,crtM, orcrtM:lpdAmutants was injected into the peritoneal cavity. These mixtures were biased (~60% mutant and ~40% wild-type) to better assess any decrease in survival due to mutations incrtM orlpdA genes. In mice infected with the mixture of the wild-type and thelpdA mutant, the fraction of the mutant decreased with time in both liver and the spleen tissues compared to its fraction in the mixture used to infect the mice (Figure 5(a)). In contrast, there was no change in the survival of thecrtM mutants as its ratio in the bacteria recovered from both liver and spleen tissues of the infected mice was similar to the ratio ofcrtM mutants in the injected mixture (Figure 5(b)). ThecrtM:lpdA double mutant also showed a decrease in its ratio in bacteria recovered from the infected liver and spleen compared to the ratio in the mixture used to infect the mice (Figure 5(c)). Survival of thecrtM:lpdA appeared to be lower even compared to thelpdA mutants, at least in liver tissues (Figures 5(b) and 5(c)).Figure 5
Survival in mice.Approximately 2X106 bacteria were injected (~60:40 ratio of mutant to wild-type). Mice were sacrificed after 8 and 24 h. Values represent average of three independent mouse tissues ± standard deviation (∗ significant atp≤.05). CFUs – colony forming units. (a)lpdA mutant:wild-type SH1000; (b)crtM mutant:wild-type SH1000; (c)crtM:lpdA mutant:wild-type SH1000.
(a) (b) (c)


## 3.1. Growth Kinetics of the Mutant and Wild-Type Strains SH1000

A comparison of the growth kinetics of the wild-type SH1000 and thecrtM andlpdA mutants showed a decreased growth rate of thelpdAmutant (Figure 1, Table 2). On the other hand, thecrtMmutant of theS. aureus strain SH1000 showed no change in growth in comparison to the wild-type (Figure 1, Table 2). The growth of thecrtM:lpdA double mutant was significantly slower than the wild-typeS. aureusas well as thelpdA individual mutant at every time point (Figure 1, Table 2). When thecrtM:lpdAwas complemented with a plasmid containing the entirecrt operon, the growth of the complemented strain remained slower like the growth of thelpdA mutant (Figure 2). However, when thecrtM:lpdAmutant was complemented with the entirebkdgene locus on plasmid pCU1, the growth of the complemented strain was restored to the level of the growth rate of the wild-typeS. aureus strain SH1000 (Figure 2).Table 2
Generation time of differentS. aureus strains.
Strain Generation time (min) SH1000 32.09 SH1000:lpdA 34.49 SH1000:crtM 32.68 SH1000:crtM:lpdA 39.51Figure 1
Growth analysis ofS. aureusstrain SH1000 and derivativelpdA andcrtM mutants.Growth was measured every 30 min (OD600) until stationary phase was reached. Values represent an average of two independent experiments.Figure 2
Growth analysis ofcrtM:lpdA double mutant and the mutant complemented with BKD orcrt gene clusterin trans.Growth was measured every 30 min (OD600) until stationary phase was reached. Values represent an average of two independent experiments.


## 3.2. Analysis of the Membrane Fatty Acid Composition of Wild-TypeS. aureus Strain SH1000 and Its DerivativecrtM,lpdA, andcrtM:lpdAMutants

Cell membrane fatty acid compositions ofS. aureuswild-type SH1000 and its isogenic mutants are shown in Table 3. In wild-typeS. aureus, the BCFAs accounts for 70.7% of the total membrane lipids. Fatty acid profile of thecrtMmutant ofS. aureus SH1000 is similar to the fatty acid profile of the parent strain with about 70% BCFAs (Table 3). ThelpdAmutant, on the other hand, showed a significant deficiency in BCFAs and represented only 35.1% of the membrane lipids (Table 3). Fatty acid profile of thecrtM:lpdAdouble mutant was similar to thelpdA individual mutant ofS. aureus SH1000 with a significantly lower BCFA level (42%) compared to their levels (70.7%) in the wild-typeS. aureus (Table 3).Table 3
Major membrane fatty acids ofS. aureus strains.
Wild-type SH1000 crtMmutant lpdAmutant crtM:lpdA C10:0 0.72 ± 0.20 0.66 ± 0.13 0.64 ± 0.40 0.67 ± 0.39  C12:0 0.60 ± 0.10 0.60 ± 0.09 0.56 ± 0.05 0.55 ± 0.04  C13:0 iso 0.25 ± 0.20 0.23 ± 0.02 0.42 ± 0.17 0.37 ± 0.17  Iso C14:0 2.60 ± 0.40 2.68 ± 0.20 8.13 ± 0.44 10.5 ± 0.33  C14:0 1.55 ± 0.40 1.61 ± 0.21 9.67 ± 1.92 7.62 ± 2.30  Iso C15:0 13.5 ± 0.90 13.59 ± 0.61 3.27 ± 0.05 3.81 ± 0.21  Anteiso C15:0 35.4 ± 2.60 35.5 ± 1.70 17.1 ± 5.93 19.4 ± 5.70  Iso C16:0 3.65 ± 0.30 3.64 ± 0.10 3.28 ± 0.76 4.55 ± 1.30  C16:0 6.20 ± 0.30 6.66 ± 0.22 16.2 ± 2.07 14.9 ± 2.34  Iso C17:0 4.14 ± 0.30 4.00 ± 0.13 0.41 ± 0.06 0.54 ± 0.14  Anteiso C17:0 7.86 ± 0.90 7.28 ± 0.57 1.65 ± 0.86 2.02 ± 1.20  C17:0 0.42 ± 0.03 0.36 ± 0.05 0.66 ± 0.10 0.27 ± 0.08  Iso C18:0 0.89 ± 0.10 0.85 ± 0.02 0.55 ± 0.19 0.66 ± 0.25  C18:0 12.0 ± 0.70 12.6 ± 0.59 20.9 ± 1.58 18.9 ± 2.23  Anteiso C19:0 1.11 ± 0.04 0.96 ± 0.05 0.35 ± 0.04 0.28 ± 0.13  C19:0 0.58 ± 0.02 0.45 ± 0.02 0.75 ± 0.11 1.21 ± 0.19  C20:0 5.44 ± 0.60 5.68 ± 0.89 10.7 ± 1.31 6.47 ± 0.92  Anteiso fatty acids 44.4 43.7 19.0 21.7  Iso fatty acids 26.3 26.2 16.1 20.4  BCFA 70.7 69.9 35.1 42.1


## 3.3. Carotenoid Levels incrtM:lpdADouble Mutant and the Complemented Strains

Inactivation of thecrtMgene resulted in a nonpigmentedS. aureus cell. In these experiments both thecrtMandcrtM:lpdAmutants were white and showed minimal carotenoid production (Figure 3). The wild-typeS. aureus SH1000 and thecrtM:lpdA double mutant complemented withcrt gene locus showed restored levels of staphyloxanthin (Figure 3). Conversely, when thecrtM:lpdA double mutant was complemented with the BKD gene cluster, there was no restoration in the mutant in terms of its ability to produce staphyloxanthin (Figure 3).Figure 3
Staphyloxanthin levels inS. aureusstrain SH1000 and isogenic mutants.Carotenoid pigments were methanol extracted from 48 h grown cultures and estimated spectrophotometrically by quantifying OD465. Values represent average of three independent experiments ± standard deviation (∗ significant atp≤.05).


## 3.4. Membrane Fluidity of thecrtM,lpdA, andcrtM:lpdAMutants and the Wild-TypeS. aureus Strain SH1000

A mutation in thelpdA gene resulted in significant decrease in the membrane fluidity of the mutant bacteria (Figure 4). Polarization value recorded for thelpdA mutant was significantly greater when compared to the polarization value for the wild-typeS. aureus suggesting a more rigid membrane. In contrast, thecrtMmutant showed polarization value slightly lower when compared with the polarization value for the wild-typeS. aureus strain SH1000 cells, but this change was not statistically significant. ThecrtM:lpdAdouble mutant was slightly more fluid than thelpdA mutant, but still significantly less fluid than the wild-typeS. aureus strain SH1000.Figure 4
Membrane fluidity ofS. aureus strain SH1000 and derivativelpdA,crtM, andcrtM:lpdA mutants. Cells from mid-exponential phase cultures were washed and incubated with DPH. Membrane fluidity was measured with a spectrofluorometer. Values represent average of three independent experiments ± standard deviation (∗ significant atp≤.05).


## 3.5. Survival oflpdA,crtM, andcrtM:lpdAMutants in Mice

In these bacterial survival studies in mice, a mixture of wild-type SH1000 with eitherlpdA,crtM, orcrtM:lpdAmutants was injected into the peritoneal cavity. These mixtures were biased (~60% mutant and ~40% wild-type) to better assess any decrease in survival due to mutations incrtM orlpdA genes. In mice infected with the mixture of the wild-type and thelpdA mutant, the fraction of the mutant decreased with time in both liver and the spleen tissues compared to its fraction in the mixture used to infect the mice (Figure 5(a)). In contrast, there was no change in the survival of thecrtM mutants as its ratio in the bacteria recovered from both liver and spleen tissues of the infected mice was similar to the ratio ofcrtM mutants in the injected mixture (Figure 5(b)). ThecrtM:lpdA double mutant also showed a decrease in its ratio in bacteria recovered from the infected liver and spleen compared to the ratio in the mixture used to infect the mice (Figure 5(c)). Survival of thecrtM:lpdA appeared to be lower even compared to thelpdA mutants, at least in liver tissues (Figures 5(b) and 5(c)).Figure 5
Survival in mice.Approximately 2X106 bacteria were injected (~60:40 ratio of mutant to wild-type). Mice were sacrificed after 8 and 24 h. Values represent average of three independent mouse tissues ± standard deviation (∗ significant atp≤.05). CFUs – colony forming units. (a)lpdA mutant:wild-type SH1000; (b)crtM mutant:wild-type SH1000; (c)crtM:lpdA mutant:wild-type SH1000.
(a) (b) (c)


## 4. Discussion

Prevalence of the bacteriumS. aureus in the environment facilitates colonization on human skin, nares, and other exposed tissues. Colonized individuals are at a greater risk of progressing to clinical infections [24–26]. Pathogenic bacteria, such asS. aureus, continuously face conditions that are hostile to their existence in the environment. Under conditions of low temperature, these pathogens are challenged to maintain the fluidity of their cellular membrane which to a large extent is regulated by the presence of branched-chain fatty acids (BCFAs). Production of BCFAs is dependent on a four-polypeptide-enzyme complex [27–29]. Inactivation of one of these polypeptide encodings genes,lpdA, resulted in significant decrease in membrane BCFAs, fluidity, growth at lower temperatures, and an increase in staphyloxanthin levels [11, 12]. Staphyloxanthin is a carotenoid pigment that likely functions as a virulence factor forS. aureus [13, 14] and at higher levels decreases membrane fluidity. Staphyloxanthin [16] also impacts the susceptibility of staphylococci to host antimicrobial peptides [16]. In this study, thelpdA mutant showed a more rigid membrane which is consistent with previous findings [11, 12]. The membrane fluidity of the staphyloxanthin deficientS. aureus, however, was almost comparable to the fluidity of the wild-typeS. aureus. In addition,S. aureus cells deficient in both, BCFAs and staphyloxanthin, showed fluidity similar tolpdA mutants.The role of staphyloxanthin in membrane fluidity ofS. aureus has been discussed in multiple reports [15–18]. A slight increase in membrane fluidity was noted in staphyloxanthin deficientcrtM mutant ofS. aureus[16]. When this mutant was complemented with the entirecrtOPQMN operonin trans resulting in a hyperpigmented strain ofS. aureus with much higher levels of staphyloxanthin, these bacteria showed a much more rigid membrane [16]. Higher level of staphyloxanthin inS. aureus makes the membrane more rigid and this has been reconfirmed in several additional studies [15, 17]. WhenS. aureus strains JE2 and SH1000 were grown in Muller-Hinton broth (MHB) and Luria-Bertani (LB) broth, they produced higher levels of BCFAs compared to when grown in brain heart infusion broth, TSB, or fetal bovine serum [10]. Both strains, JE2 and SH1000, that produced higher BCFA in MHB, also produced higher levels of staphyloxanthin. However, the LB broth grown SH1000 and JE2 cells, with higher BCFA levels, showed no increase in their staphyloxanthin content [10]. Similarly, the MHB grown cells showed a higher polarization value in membrane fluidity experiments suggesting a rigid membrane but the LB broth grown cells showed no such decrease in fluidity [10]. The authors hypothesized that the increase in membrane fluidity was counterbalanced by increasing the level of staphyloxanthin in these bacteria [10]. However, this does not explain why elevated levels of staphyloxanthin was not seen in all of the cultures with increased BCFAs.S. aureus cells that lack a functional BKD enzyme, which is believed to be the main bacterial enzyme responsible for BCFA synthesis, showed higher levels of staphyloxanthin [12]. Overall, BCFA appears to be the main regulator of membrane fluidity inS. aureus.In the analysis of the fatty acids in the cellular membrane, thelpdAmutant ofS. aureus showed a decreased production of BCFAs, which is consistent with prior reports [11, 12]. On the other hand, the fatty acid composition of thecrtM mutant was similar to the membrane fatty acids of the wild-typeS. aureus. Fatty acid composition of thecrtM:lpdAdouble mutant was deficient in BCFAs and similar to the fatty acid composition of thelpdA mutants. The findings suggest that there is no correlation between staphyloxanthin and BCFA levels inS. aureuswhich is consistent with prior reports [16, 17].Survival ofcrtM andlpdA mutants was also investigated underin vivo conditions in an intraperitoneal mouse infection model. We found that thelpdA mutants and thecrtM:lpdAdouble mutants had significantly reduced survival and were cleared from infected mice at a faster rate compared to the wild-typeS. aureus. Mutation inlpdAgene has previously been shown to have a decreased survival in mice [11]. Although, thelpdA mutants showed slowerin vitro growth, it is unlikely to be the sole reason for the poor recovery of these mutant bacteria from the mouse tissues. Similar CFUs were injected in case of thelpdA,crtM, orcrtM:lpdA double mutants in combination with the wild-type bacteria. In addition, the recovery of total tissue CFUs was comparable in case of the three mixtures that were injected in mice. These experiments were concluded within 24 h and during this timein vivo, the bacteria faced a hostile environment. While a lower growth rate of thelpdA mutant might be a contributing factor, it is more likely that the decrease in membrane BCFAs puts the cells at a disadvantage underin vivo conditions. There was no change in the survivability of thecrtM mutants in our investigations.However, previous studies have shown that the lack of staphyloxanthin increases the vulnerability ofS. aureus cells to neutrophil killing [13, 30]. In that investigation, lack of staphyloxanthin reduced the virulence potential ofS. aureus in a mouse skin abscess model [13].The potential of staphyloxanthin as a drug target has recently been investigated [19]. When Naftifine was used to inhibit staphyloxanthin production (by inhibiting the activity of the enzyme encoded bycrtN) in mice infected with methicillin-resistant staphylococci, these bacteria showed significantly reduced bacterial counts in kidneys and hearts of treated mice compared to untreated mice [19]. In contrast, the liver of the mice showed no change in bacterial load in Naftifine treated mice vs nontreated mice [19]. ThecrtMmutant used in this study showed no significant reduction in survival underin vivo conditions. This seems in contrast to the previous findings [13], and it may be due the strain used in this study (which is a methicillin-sensitive strain vs methicillin-resistant strain), the type of infection model (intraperitoneal vs skin abscess model), and the infected tissues that were analyzed. Nonetheless, thecrtM:lpdAmutant showed an even greater decrease in survival compared to the individualcrtMorlpdAmutants.


## 5. Conclusions

In summary, a mutation in thelpdAgene decreases the growth rate, membrane fluidity, and virulence ofS. aureus. Mutation in thecrtMgene eliminates pigment production, but this lack of staphyloxanthin has little to no effect on growth rate, membrane fluidity, or virulence ofS. aureusstrain SH1000. A double mutation incrtM andlpdA genes showed a much greater growth defectin vitro. These double mutants also showed reducedin vivo survival suggesting that the simultaneous targeting of the BCFA and staphyloxanthin metabolic pathways inS. aureus may be an attractive strategy to control infections caused by this pathogen.



---

*Source: 2603435-2019-01-22.xml*