Transcriptome profiling of mastitis-specialized Staphylococcus aureus reveals the impact of low-oxygenation on the regulation of unique pawthways after internalization into bovine mammary alveolar cell-T (MAC-T)

Background Mastitis-specialized lineages of Staphylococcus aureus are important pathogens in the dairy industry. The molecular mechanisms underlying host- and organ-specialization in these lineages are still not fully understood. Recent findings suggested that differential expression of genes may have contributed to the evolution of strains with enhanced virulence. However, studies on gene expressions under key intra-mammary conditions are quite limited for mastitis S. aureus . The purpose of the study was to investigate the influence of low oxygen levels on the transcriptome profiles of bovine matitis S. aureus , using high-throughput whole genome qRT-PCR.Results Results showed that under normal oxygenation, a mastitis-isolate expressed subsets of genes for adaptation, environmental-sensing, and binding including merR, sigB , vraS , yycG/yycF , araC , and tetR . In addition, coupling of fermentative metabolism to virulence was indicated by accumulated transcripts for catabolite control protein A ( ccpA) and pentose-monophosphate operon and depleted transcripts for tricaroxylic acid cycle. Furthermore, sarU mediated agr activation was evidented by transcripts for toxins, adaptation, and in-vivo viability factors as staphopains and gntR operon. On the other hand, reduced oxygenation increased transcription of fibrinogen-binding genes, isd- operon, and sdrH showing aggressive adherence phenotype. While normal oxygenation produced gene activities for quick and aggressive responses, low-oxygenation induced phenotypes for persistence, binding, and metabolic inactivity.Conclusion Significant differences in the transcriptional profiles were observed for mammary alveolar cell-T (MAC-T) internalized S. aureus under low oxygen levels compared to that at normal levels. This indicated that low oxygen is an important key mammary factor aureus . These findings will help in understanding the effect of oxygen on the differentiation and evolution of intramammary S. aureus cDNA

that influence transcriptome profiles of intra-mammary-specific phenotypes of S. aureus . These findings will help in understanding the effect of oxygen on the differentiation and evolution of intramammary S. aureus .

Introduction
Mastitis is a most costly disease for the dairy industry. It affects milk production, animal welfare, and food safety. For example, mastitis costed $662 Canadian dollars per milking cow per year for a typical Canadian dairy farm [1]. Another study conducted in Finnish dairy has identified Staphylococcus aureus as the most important pathogen for culling dairy cows [2].
Staphylococcus aureus is one of the most important contagious mastitis pathogens in dairy cattle. Until 1995, a single common clone (electrophoretic type, ET3) was responsible for a majority of bovine mastitis cases [3]. Since then, more virulent subclonal populations were identified that showed hypersusceptiblity to acquisition of resistance [4,5]. While it is known that acquired resistance increases epidemicity, it does not significantly alter the inherient adaptive evolution of virulence per se. In addition, the predominant strains of this species that cause bovine mastitis in different geographic regions continue to be intra-mammary specialized and methicillin-sensitive S. aureus (MSSA) [6].
Despite enormous efforts, the regulatory process that control the rapid evolution and emergence of specialized S. aureus strains in different host tissues is still elusive. Fitness in host tissues and organs through processes of cellular differentiation and/or acquisition of lineage-specific trait(s) has been suggested [7,8]. However, less is known about how the host factors, such as low oxygen levels during mastitis, induce specific regulatory pathways in the pathogen, which will lead to selection of mastitis-specific S. aureus in the mammary gland. Using Burkholderia dolosa Lieberman et al., (2011) [9] were able to identify oxygen-dependent adaptive evolutions affecting important pathogenic traits during human lung infections. In the un-inflammed bovine mammary gland, oxygen levels are around 23 mmHg.
However, in S. aureus infected mammary gland, that level has been found further reduced to only 1.3 mmHg [10]. Therefore, the objectives of this study were to investigate the whole-genome transcriptional profiling of an mammary-specialzed S. aureus isolate after in vitro internalization into mammary epithelial cells under normal and low oxygen levels.

Bacterial strains and culture preparation
In these experiments, we used a S. aureus isolate from a dominant mastitis clone. This S. aureus isolate was identified as Publsed Field Gel Electrophoresis (PFGE) type A (A7 and A3) corresponding to clfA subtype Q, identical to the sequenced RF122 isolate [11]. It was used in mammary alveolar cell-T (MAC-T) invasions under normal (incubator with 5% CO 2 ) and reduced oxygen (5% O 2 ) conditions. The reduced oxygen condition was maintained in a Modular Incubator Chamber as explained below. The isolate was stored at -80°C without subjecting to extensive subculturing to avoid genetic changes. Innoculum cultures were prepared according to Bayles et al., (1998) [12] with some modifications. Overnight cultures (grown in the invasion medium, which was the cell culture growth medium without antibiotics and Fetal Bovine Serum (FBS), as defined below) were centrifuged and the pellet was washed once with sterile phosphate-buffered saline (pH 7.2) and resuspended in 10 ml of the invasion medium to give a density of 10 10 CFU ml -1 . Serial dilutions of this were prepared and 1 ml/well of the 10 2 dilution was used to inoculate MAC-T cell monolayers at the multiplicity of infections of 100 (MOI 100).

Cell culture
A bovine mammary epithelial cell line designated MAC-T [13]  For normal oxygenation, cells were cultured in the incubator with 5% CO 2 . To ensure adequate amount of oxygen flux into cells, culture plates with wide-surface, flatbottom, and shallow wells were used that were inoculated with a thin layer of media (1ml) with frequent swirling of plates. These measures have been found necessary, as the oxygen does not readily diffuse into culture media [14]. For reduced oxygenation, the Modular Incubator Chamber model 101 (Billups-Rothenberg, Inc, California, USA) was used to create lower limits of microaerophilic conditions. To achieve low oxygen level, 5% oxygen in the Modular Incubator Chamber (37.5 mm Hg) was used. For each experiment in these chambers, two 6-well tissue culture plates were simultaneously incubated under the same condition; one with cocultures and the other with free bacteria without host cells to act as control. The experiment was repeated three times (3x) each under normal and reduced conditions. This design aimed to mimick the intra-mammary oxygen levels during mastitis.
Approximately 16 h prior to experiment the cell growth medium was replaced with 1 ml of the invasion medium. The morning of the experiment, the medium was removed and monolyayers were washed once with the invasion medium. The MAC-T cell plates were then inoculated with S. aureus in the invasion medium at the MOI 100 and incubated at 37°C under normal oxygenation or reduced oxygenation.
Plates containing only free bacteria in the invasion medium without host cells were used as the control for both oxygenation conditions After 1 h, supernatants of the cocultures were removed and monolayers washed three times with the invasion medium containing lysostaphin (10 µg ml -1 ; Sigma) to kill extracellular bacteria [16,17]. Plates were then re-washed with the invasion medium before incubating in the fresh invasion medium containing 100µg /ml gentamicin (Invitrogen Inc.) for 8h. water. Nine ml of diluted cDNA and 15ml of Roche 2x SYBR Green master were mixed. Eight µl of cDNA/mastermix was aliquoted into 384-well plates. Primers were added to cDNA master mix to a final volume of 10µl. Plates were sealed with qRT-PCR tape and stored in the -80 until use. Plates were run in a Roche LifghtCycler480 (LC480) RealTime PCR system 384-plate format for gene-expression, interfaced with robotics to create an automated high-throughput. qRT-PCR cycles were as follow: Step 1: 5 minutes at 95°C.

Transcriptome profiles of mastitis S. aureus isolate internalized in mammary cells at normal O 2 levels
Transcript levels of genes that were either up-or down-regulated in response to normal oxygen condition in incubator were measured. In the up-regulated category, elevated levels of transcripts were produced in transport and binding proteins such as detoxification and adaptation genes mmpL efflux pumps like norG, pnbA (paranitrobenzyl esterase), betA and betB (betaine hydrolysis), and camS as well as in cellular processes. Inreased transcriptional activities were also seen in specific subsets of regulatory and pathogenesis genes such as agr signaling molecules, sarU, vraSR, merR, araC, gntR, tetR, sspB2, superantigens, and proteases ( Figure 1 and Table 1). Interestingly, transcript levels for genes in glycolysis, fermentation, and anaerobic pathways were higher and these genes included fructosebisphosphatase, L-lactate dehydrogenase (ldh1), and fructose-1,6-bisphosphate aldolase (fdaB). Genes for aldehyde dehydrogenase (aldA2), bifunctional acetaldehyde-CoA/alcohol dehydrogenase, alpha-acetolactate decarboxylase (budA2), pyruvate-formate-lyase-activating enzyme (pflA, pflB), formate acetyltransferase genes, and phenol soluble modulins were also expressed.
Significant increases also occurred in transcript levels for cell envelope, energy, DNA metabolisms, and other regulatory functions. On the other hand, transcripts for major biosynthetic and central intermediary metabolism processes were downregulated. These included L-lactate dehydrogenase (ldh2) locus (SACOL261) and PTS components IIABC (locus SACOL2552), fibronectin binding protein (FnbP) (SAB1289c) and nuoF (NADH dehydrogenase subunit 5) ( Figure 2 and Table 2).

Transcriptome profiles of mastitis S. aureus internalized into mammary cells at reduced oxygen condition.
Under reduced oxygen, up-regulated and down-regulated genes were also observed.
As shown in Figure 3, the majority of up-regulated gene activities were for surface and envelope proteins involved in transport, binding, and cellular processes. For example, transcript levels of cation-induced transport processes i.e., sodium-, iron-, and ammonium-mediated transport, as well as surface proteins such as fibrinogen binding related proteins, iron-regulated surface determinant (Isd) protein genes isdA, isdC, isdH, and serine aspirate dipeptide repeat protein genes (sdrH) were elevated. In addition, genes for adaptation proteins such as phenol soluble modulins, aerolysins, superantigen were also transcribed ( Table 3). The down regulated genes under the reduced oxygen condition included some components of glycolytic and fermentative pathways such as gntR, fructose and sucrose metabolic genes, and many hypothetical genes (21.4%) ( Table 4). In addition, genes related to purines, pyrimidines, nucleotides, and nucleosides were mostly suppressed ( Figure   4).

Discussion
In this study, we have identified specific genes and pathways that were expressed At the reduced oxygen level, (Figure 3, Table 3) fibrinogen binding, iron-regulated surface determinant genes isdA, isdC, and isdH, and serine aspirate dipeptide repeat (sdrH) were expressed. This result is consistent with their induction by iron depletion [28]. Interestingly, the unique suppression of genes for metabolism of purines, pyrimidines, nucleotides, and nucleotides would reflect limited activities in bacteria, since these molecules are well-known sources of energy or substrates for other metabolisms. The ATP or GTP are used for protein synthesis, while UTP are needed for activating glucose and galactose. Similarly, CTP is essential for lipid metabolism, while coenzyme A, or nucleotides are required for the synthesis of nucleic acids. This finding is supported by significant downregulation in metabolic pathways ( Figure 4, Table 4).

Conclusion
We have conducted transcriptome analysis of internalized mastitis-Staphylococcus aureus isolate at low and normal oxygen tentions. Data indicated that at low oxygen, unique set of transcriptome profiles were produced compared to normal oxygen. Transcripts of fermentation and PMP pathways even at normal oxygen level, catabolite control repression, and virulence were strikingly co-accumulated. While

Availability of data and material
All relevant data are within this paper. Additional tables of full data are also provided.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by funding from a discovery grant from Natural Science and Engineering Research Council of Canada given to Dr Xin Zhao and in a collaboration with JCVI and Drs SP, MBJ, and RB.