Alberto Alberti,2 Marco Pittau,2 Tonina Roggio,1and Sergio Uzzau1,5
Porto Conte Ricerche Srl, Tramariglio, Alghero, Italy1; Dipartimento di Patologia e Clinica Veterinaria, Universita di Sassari,Sassari, Italy2; Istituto Zooprofilattico Sperimentale della Sardegna G. Pegreffi, Sassari, Italy3; Biosistema Scrl, Sassari,
Italy4; and Dipartimento di Scienze Biomediche, Universita di Sassari, Sassari, Italy5
Received 11 January 2011/Returned for modification 26 February 2011/Accepted 1 June 2011
Milk fat globules (MFGs) are vesicles released in milk as fat droplets surrounded by the endoplasmicreticulum and apical cell membranes. During formation and apocrine secretion by lactocytes, various amountsof cytoplasmic crescents remain trapped within the released vesicle, making MFGs a natural samplingmechanism of the lactating cell contents. With the aim of investigating the events occurring in the mammaryepithelium during bacterial infection, the MFG proteome was characterized by two-dimensional difference gelelectrophoresis (2-D DIGE), SDS-PAGE followed by shotgun liquid chromatography-tandem mass spectrom-etry (GeLC-MS/MS), label-free quantification by the normalized spectral abundance factor (NSAF) approach,Western blotting, and pathway analysis, using sheep naturally infected by Mycoplasma agalactiae. A number ofprotein classes were found to increase in MFGs upon infection, including proteins involved in inflammationand host defense, cortical cytoskeleton proteins, heat shock proteins, and proteins related to oxidative stress.Conversely, a strikingly lower abundance was observed for proteins devoted to MFG metabolism and secretion.To our knowledge, this is the first report describing proteomic changes occurring in MFGs during sheepinfectious mastitis. The results presented here offer new insights into the in vivo response of mammaryepithelial cells to bacterial infection and open the way to the discovery of protein biomarkers for monitoringclinical and subclinical mastitis.
Mycoplasma agalactiae is the causal agent of contagious aga-lactia (CA), one of the most serious infectious diseases affect-ing small ruminants worldwide (11, 22, 34). The main clinicalsign of CA in acutely infected flocks is the alteration of milkconsistency in lactating ewes, with a decline and subsequentfailure of milk production as a result of interstitial mastitis.Also, arthritis and keratoconjunctivitis can affect about 5 to10% of infected individuals (4). In areas where CA is endemic,however, subacute and chronic infections are by far the mostfrequent occurrences (10, 11), making control and eradicationof this pathogen extremely difficult. In fact, acute mastitis sub-sequent to mammary gland infection often progresses to sub-acute or chronic disease, during the course of which the patho-gen is shed in milk for extended periods; after clinicalresolution of the disease, mycoplasmas continue to be shed andanimals become asymptomatic carriers (4). Eradication planshave been in place for decades in several countries, but the
disease persists in many areas, where it is still responsible forimportant economic losses.
Although CA is widely described clinically, the molecularpathogenesis of CA is not well understood, and the host-patho-gen interplay during natural mycoplasma infection has yet tobe elucidated. In general, M. agalactiae affects the mammaryglands, joints, eyes, ears, and respiratory tract, causing inflam-mation with different degrees of severity. The microorganism isbelieved to be unable to penetrate cells, although it is known toadhere tightly to the colonized epithelium (34, 37). Despiterecent in-depth genomic and proteomic analyses (7, 27, 40),which have led to fundamental insights into its genomic andproteomic composition, very few proteins of M. agalactiae havebeen proved to be virulence determinants. These include afamily of variable surface proteins, named Vpma proteins (16),the immunodominant adhesin P40 (14), and P48, a homologueof the macrophage-activating lipopeptide (MALP) of M. fer-mentans (36).
The lack of knowledge concerning virulence mechanismsmight also be due to the scarcity of data on alterations occur-ring in the host cell counterpart. To date, there are no pro-teomic or genomic studies aimed at investigating the responseof sheep tissues to M. agalactiae, and expression levels of low-abundance milk proteins have seldom been assessed duringmastitis. The milk proteome has been investigated in naturally
* Corresponding author. Mailing address: Porto Conte Ricerche Srl,S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Algh-ero SS, Italy. Phone: 39-79-998-526. Fax: 39-79-998-567. E-mail: [email protected].
‡ Supplemental material for this article may be found at http://iai.asm.org/.
† M.F.A. and S.P. contributed equally to this work.� Published ahead of print on 20 June 2011.
and experimentally infected cows with signs of mastitis (5, 6,19, 41); nevertheless, the response of the mammary gland tonatural mycoplasma infections has never been subjected to adedicated study. To the best of our knowledge, just one studyevaluated the local immune response of the goat mammarygland in an experimental M. agalactiae infection (9).
Milk fat globules (MFGs) are released from the lactatingcell as a result of an apocrine secretion mechanism leading tothe formation of fat droplets surrounded by the endoplasmicreticulum (ER) membrane and by the apical cell membrane onthe external surface (17). Cytoplasmic crescents are oftentrapped between these membrane layers (17), making MFGs anatural mechanism for sampling the lactating cell in vivo. Inthis way, the molecular content of MFGs might be exploited tostudy the biology of the lactating cell and to evaluate thealterations occurring in vivo under pathological conditions.Indeed, several proteins involved in host defense have beenidentified in bovine milk during natural and experimentallyinduced mastitis, and some of these have been associated withMFGs (19, 41, 42). Moreover, MFGs display intriguing simi-larities with exosomes, small secretory vesicles released by sev-eral tissues and involved in manifold functions, including im-munomodulatory activity (33, 39, 49). As a further advantage,the investigation of a purified subproteome directly derivedfrom the lactating cell reduces sample complexity and uncoversthe deep proteome represented by low-abundance proteins,overcoming the problems generated by the massive amounts ofcaseins and whey proteins present in milk.
Recently, we accomplished an in-depth proteomic charac-terization of sheep MFG proteins (MFGPs) under physiolog-ical conditions, revealing a complex but highly reproducibleprotein profile for midlactation ewes (31). Here we compara-tively investigated the proteomic profiles of MFGPs in sheepmilk samples collected from CA-affected and CA-free flocks.Protein expression profiles were evaluated by means of two-dimensional difference gel electrophoresis (2-D DIGE) andSDS-PAGE followed by shotgun liquid chromatography-tan-dem mass spectrometry (GeLC-MS/MS), and protein abun-dance data were compared to the healthy sheep MFGP profile.Differentially expressed proteins were identified and charac-terized by gene ontology (GO) and pathway analyses.
MATERIALS AND METHODS
Animals and samples. For this study, frozen 50-ml milk samples belonging toa total of 7 CA-affected flocks were retrieved retrospectively from the IstitutoZooprofilattico Sperimentale della Sardegna (IZS; a public animal health insti-tution), with samples classified as positive for M. agalactiae culture or as micro-biologically negative (n � 12 and n � 15, respectively; total number of samples �27), and were used for proteomic investigations. Samples from an M. agalactiae-free flock were also collected as negative controls (n � 3; total number ofsamples � 30). All milk samples were retrieved among those collected by IZS aspart of the control and eradication program established by the Sardinian Re-gional Government. According to this program, sheep belonging to flocks whereCA outbreaks have occurred or are suspected are subjected to clinical examina-tion by the IZS veterinary personnel, and 50 ml of mixed milk from bothhalf-udders is collected as described previously (24). Samples are refrigeratedand subjected to microbiological examination at the IZS laboratory within 24 hof collection (24). For microbiological cultures, 10 �l of milk is seeded in 5%sheep blood agar and incubated at 37°C for 24 to 48 h. For mycoplasma culture,10 �l of milk is seeded in Hayflick agar and incubated in a wet chamber for upto 7 days. Upon growth of fried-egg colonies, isolates are cloned and identifiedby PCR (45). For this study, only milk samples which were positive for M.agalactiae culture and microbiologically negative for other microbial pathogens
were selected and retrieved. Once transported to the proteomics laboratory, allmilk samples (n � 30) were thawed and tested by PCR for the presence of M.agalactiae DNA (7, 45).
Extraction of sheep MFGPs. MFGPs were extracted as described previously(31) from the same sheep milk samples examined by bacterial culture andsubjected to whole-milk PCR for detection of M. agalactiae. Briefly, milk sampleswere centrifuged to obtain the cream fraction containing MFGs, and the creamwas washed twice in phosphate-buffered saline and once in triple-distilled water.The fat globules were then crystallized at 4°C overnight, homogenized in triple-distilled water with a TissueLyser mechanical homogenizer (Qiagen, Hilden,Germany), and then warmed to melt the fat. After centrifugation, the pellet wasresuspended in water and the protein concentration was determined with a 2-DQuant kit (GE Healthcare, Uppsala, Sweden). Samples were stored at �20°Cprior to analysis.
SDS-PAGE and Western immunoblotting. MFGP samples (n � 21) wereresuspended in Laemmli buffer (21), boiled, loaded into precast TGX acrylamidegels (Bio-Rad Laboratories, Hercules, CA), subjected to electrophoresis (1), andstained with Coomassie blue (8). Western immunoblotting was performed asdescribed previously (1), using the following antisera: monoclonal anti-actin(clone AC-40) antibody, monoclonal anti-�-tubulin (clone B-5-1-2) antibody,anti-S100A9 rabbit antibody, anti-cathelicidin rabbit antibody, monoclonal anti-S100A11 (clone 2F4) antibody (Sigma-Aldrich, St. Louis, MO), and a rabbithyperimmune serum raised against recombinant M. agalactiae P48 (M. agalactiaerP48) (36).
2-D DIGE. Sixty-microgram protein samples extracted from triplicate samplesof mycoplasma-negative milk samples (uninfected) and mycoplasma-positivemilk samples (infected) were labeled with 400 pmol N-hydroxysuccinimidyl esterof cyanine dyes Cy3 and Cy2, respectively (GE Healthcare), as indicated by themanufacturer. The Cy5 cyanine dye was used to label a pooled sample compris-ing equal amounts of each of the specimens in the study (uninfected and in-fected), which served as the internal pooled standard. The labeled protein sam-ples and the internal pooled standard were mixed in suitable combinations,brought to the final rehydration volume with IPG buffer (GE Healthcare) andDestreak rehydration solution (GE Healthcare), and applied to 24-cm IPG strips(pH 3 to 11, nonlinear; GE Healthcare) by passive rehydration overnight at roomtemperature (see Table S1 in the supplemental material). Rehydrated strips werethen run together in an IPGphor device equipped with an Ettan IPGphor 3loading manifold (GE Healthcare) at 20°C for a total of about 90,000 V-h. Afterisoelectric focusing (IEF), the strips were equilibrated (6), subjected to second-dimension SDS-PAGE, and digitalized as described previously (44). The imageswere analyzed with a DeCyder batch processor and differential in-gel analysis(DIA) modules (GE Healthcare). Statistical analysis of protein level changes wasperformed with the DeCyder BVA (biological variation analysis; v.6.5) module.The results related to uninfected and infected samples were compared andstatistically evaluated by one-way analysis of variance (ANOVA) with theDeCyder BVA module, applying the false discovery rate (FDR) to minimize thenumber of false-positive results. Protein spots with statistically significant varia-tion (P � 0.05), showing a difference in volume of �2-fold, were selected asdifferentially expressed. Cluster analysis and visualizations were performed usingthe DeCyder EDA (extended data analysis) module.
MALDI-TOF MS. For protein identification, preparative 2-D PAGE gels wereset up by overnight rehydration loading of 300 �g of protein extract into pH 3 to11 NL 24-cm IPG strips. Strips were then focused and subjected to second-dimension electrophoresis as described above. All blue molecular weight mark-ers (Bio-Rad Laboratories) were also loaded on an electrode wick and runtogether with the isoelectrofocused strips. After electrophoresis, the gel slab wassubjected to colloidal Coomassie staining (8). Visible protein spots of interestwere excised manually from the gels, and mass spectra were recorded on amatrix-assisted laser desorption ionization–time of flight mass spectrometry(MALDI-TOF MS) instrument (MALDI micro MX; Waters, Manchester,United Kingdom) as described previously (2, 7, 44). Raw data, reported asmonoisotopic masses, were then introduced into our in-house Mascot peptidemass fingerprinting software (version 2.2; Matrix Science, Boston, MA) and usedfor protein identification.
GeLC-MS/MS and pathway analysis. Proteins (30 �g) were resolved by SDS-PAGE. After staining by colloidal Coomassie blue (8), the gel lane was cut into30 slices and subjected to in-gel tryptic digestion as described previously (1).Briefly, the gel slices were destained, washed, reduced, alkylated, and digestedwith trypsin; peptides were extracted, dried, and resuspended as described pre-viously (1, 7, 31, 43). LC-MS/MS analyses were performed on a quadrapole TOF(Q-TOF) hybrid mass spectrometer equipped with a nano-lock Z-spray sourceand coupled online with a CapLC capillary chromatography system (Waters) asdescribed previously (31, 43). In this case, protein identification was carried out
in the NCBI database, using the Mammalia (mammals) and Mycoplasma taxon-omies with the following search parameters: peptide tolerance, 30 ppm; MS/MStolerance, 0.4 Da; charge state, �2 and �3; enzyme, trypsin; and up to twomissed cleavages. For false-positive analysis, a decoy search was performedautomatically by choosing the decoy checkbox on the search form. Mascot resultswere parsed using the IRMa toolbox (version 1.26.1) (13) with the followinginclusion criteria in order to keep an FDR value of �2% for each analysis:protein significance threshold, P value of �0.01; peptide rank, 1; and peptide ionscore cutoff, 20. Unique peptides (UP), spectral counts (SpC), and exponentiallymodified protein abundance index (emPAI) values were reported as calculatedby IRMa (13). Skin keratins were excluded from the final protein list. Proteinswith similar peptides which could not be differentiated based on MS/MS analysisalone were grouped (38). SpC values were used as a semiquantitative parameterfor estimating protein abundance and comparing the expression of the sameprotein among different samples, as described previously (31, 43). The SpC logratio (RSC) and normalized spectral abundance factor (NSAF) were calculatedaccording to the methods of Old et al. (28) and Zybailov et al. (54), respectively,considering for each identified protein the average SpC value among biologicalreplicates. NSAF values were used to compare expression of diverse proteinsamong samples. NSAF was calculated as described previously (31, 53).
GO assignments were carried out using online DAVID bioinformatic re-sources (version 6.7) (12, 18). NSAF comparisons among classes were plottedusing Microsoft Excel, and the statistical significance of differences in proteinexpression levels among the groups compared during the study was assessed bya two-tailed t test with a 95% confidence level. The beta-binomial test wasperformed to identify differentially expressed proteins according to the methodof Pham et al. (30), using the software kindly provided by the authors. With theaim of increasing the stringency of this analysis, proteins identified with less thanone SpC in at least one replicate or with fewer than two SpCs in more than onereplicate of the subgroup in which they were more expressed were excluded fromthe list. The list of protein identifications (IDs) with P values of �0.05, togetherwith their respective RSC values, was imported into the online software packageIPA (version 8.7; Ingenuity Systems, Redwood City, CA), and network analyseswere performed with thresholds of 1.5 for RSC and 0.05 for P value. SheepUniProt IDs were replaced with the UniProt IDs for the closest human proteinequivalents in order to enable the best exploitation of the knowledge-based IPAsoftware (version 8.8; updated 13 November 2010). To determine the biologicalprocesses, functions, pathways, and molecular networks most significantly alteredduring infection by M. agalactiae, both over- and underrepresented proteins weredefined as value parameters, all identifier types and data sources were selectedin order to access all available information in the IPA database, and both directand indirect relationships were considered.
RESULTS
Alterations in total protein profile of MFGPs in sheep in-fected by M. agalactiae. In a previous study, the proteomicprofile of sheep MFGPs was characterized under physiologicalconditions, revealing a high interindividual reproducibility ofthe MFGP expression profile for healthy sheep of the dairybreed Sarda (31). In order to evaluate possible changes inMFGPs during mastitis, 27 sheep milk samples from 7 differentflocks affected by CA were retrieved from IZS, a public animalhealth institution, and 3 sheep milk samples were collected ascontrols from a certified CA-free herd. At the beginning of thestudy, all samples were subjected to whole-milk PCR to detectthe presence of M. agalactiae DNA and to extraction ofMFGPs, which were obtained successfully from 21 samples(Table 1). MFGP expression profiles were initially evaluated inrepresentative milk samples from sheep with M. agalactiae-positive cultures, from sheep with M. agalactiae-negative cul-tures but belonging to the same CA-affected flock, and fromsheep belonging to a CA-free flock (Fig. 1, left panel). Dra-matic alterations were evident in the SDS-PAGE profiles ofMFGPs from sheep with M. agalactiae-positive cultures (lanes1 and 2) compared to those for MFGPs from bacteriologicallynegative sheep from the same flock (lanes 3 and 4). However,
slight differences could also be observed in the latter samplescompared to MFGPs of sheep from the CA-free flock (lanes 5and 6). In order to investigate the presence of mycoplasmaproteins in culturally negative animals, the same samples weresubjected to Western immunoblotting (WB) with antibodiesdirected against P48, an immunodominant protein of M. aga-lactiae (36). In fact, since MFG membranes (MFGMs) arederived directly from the lactocyte apical membrane, bacterialproteins could remain associated with this cellular fraction andbe shed in milk during chronic infections. Should this be true,bacteria or proteins adhered to MFGMs would be enrichedand their detection by means of immunological techniqueswould be enhanced. The results are shown in Fig. 1 (rightpanel). As expected, samples 1 and 2 (culturally positive sam-ples) showed clearly positive signals, while samples 5 and 6(samples from the M. agalactiae-free flock) were negative.However, samples 3 and 4, obtained from sheep which wereculturally negative but belonged to the flock infected by M.agalactiae, showed a clearly visible band (sample 4) or a faintbut visible signal (sample 3), highlighting the presence of bac-terial proteins in these culturally negative samples. Moreover,this result indicates the ability of MFGs to enrich diagnosticallyrelevant proteins of the infecting bacterial pathogen, since milk
TABLE 1. Summary of sheep milk samples used in this worka
SampleID Flock PCR
resultCultureresult
Presence offat ring
1 A � � Y2 A � � Y3 A � � Y4 A � � Y5 A � � Y6 A � � Y7 A � � N8 A � � N9 B � � Y10 B � � Y
11 B � � Y12 B � � N13 B � � N14 B � � N15 C � � Y16 C � � Y17 C � � Y18 C � � N19 D � � Y20 D � � N
21 E � � Y22 E � � Y23 E � � Y24 E � � N25 E � � N26 F � � Y27 G � � Y28 K � � Y29 K � � Y30 K � � Y
30 8 25 12 21
a Results obtained by culture and milk PCR for detection of M. agalactiae arereported. The presence (Y) or absence (N) of a milk fat ring upon centrifugationis also indicated. Samples subjected to 2-D DIGE and GeLC-MS/MS are indi-cated in bold.
VOL. 79, 2011 SHEEP MFG PROTEOMICS IN M. AGALACTIAE INFECTION 3835
samples from the same animals produced a negative resultwhen tested with the same antibody (data not shown).
2-D DIGE characterization of sheep MFGPs during M. aga-lactiae infection. In view of the preliminary observations bySDS-PAGE, a 2-D DIGE study was performed with the aim ofcharacterizing, both qualitatively and quantitatively, the differ-ences in MFGPs of infected and uninfected animals. There-fore, three samples from the CA-free flock and three samplesfrom the CA-affected flock, one of which was negative forbacterial culture but showed WB positivity for P48 (C�/WB�),were subjected to a 2-D DIGE study. Figure 2 summarizes theobtained results. A representative 2-D DIGE image comparingsignals generated by a negative sample (C�/WB�) (green sig-nal) and by a culturally M. agalactiae-positive sample (C�/WB�) (blue signal) is shown, together with the six individual2-D maps used for image analysis and statistical analysis.
Analysis of the 2-D DIGE images with DeCyder software(GE Healthcare) revealed statistically significant differences inabundance for numerous spots, 28 of which were successfullyidentified by MS (Fig. 2; Table 2). The proteins found to beincreased in positive samples were involved mostly in mem-brane and vesicular trafficking (30%), such as annexins, actin,and myosin; in immune function, inflammation, and host de-fense (30%), such as S100 proteins, cathelicidins, and antimi-crobial peptides; in protein synthesis and folding (25%), suchas heat shock proteins (HSPs); and in enzymatic activity(15%), such as the mitochondrial superoxide dismutase andprotein disulfide isomerase. In contrast, proteins found to bedecreased were involved almost exclusively in fat transport/metabolism and in MFG secretion, including butyrophilin, lac-tadherin, adipophilin, and xanthine dehydrogenase/oxidase,with only a minimal fraction being represented by milk pro-teins.
Multivariate analysis based on principle component analysis(PCA) was performed on expression data to assess globalchanges in the MFGP profiles for infected sheep (C�/WB�
and C�/WB�) compared to uninfected sheep (C�/WB�). PCAallows for grouping of samples with overall similar expressioncharacteristics and for identification of proteins which are re-sponsible for the differences between groups. PCA with the
differentially abundant proteins revealed a clear separation ofpositive and negative samples into two groups (Fig. 3, toppanel, red and purple groups) in the score plot. Interestingly,the sample with culture negativity and WB positivity (C�/WB�) clustered separately from the other sample groups (Fig.3, top panel, green group). A heat map was also generated inorder to compare protein expression patterns within the threeclasses: uninfected (C�/WB�), infected (C�/WB�), and in-fected but culture negative (C�/WB�). The results obtained,illustrated in Fig. 3, bottom panel, further highlight the differ-ences in expression existing between infected and noninfectedindividuals and the intermediate status of the C�/WB�
sample.GeLC-MS/MS analysis of sheep MFGPs during M. agalac-
tiae infection. Proteome coverage was subsequently increasedby means of GeLC-MS/MS analysis. This approach overcomessome of the limitations suffered by 2-D PAGE in analysis ofmembrane proteins and liposoluble proteins. Moreover, bymeans of recent advancements in label-free quantification, theability to perform a relative evaluation of protein expressionamong samples is maintained (30, 52). To this extent, MFGPsextracted from the two M. agalactiae-positive samples exam-ined by 2-D DIGE were also subjected to GeLC-MS/MS anal-ysis. In total, 185 unique proteins were identified, subjected tolabel-free relative quantification of protein expression bymeans of the spectral counting approach, and normalized bymeans of the NSAF (28, 54) as described previously (31, 43)(see Table S2 in the supplemental material). A protein ID andabundance database was generated for healthy sheep MFGs(31). Building on this existing database, a beta-binomial testwas applied to spectral counting data with the purpose ofidentifying proteins with statistically significant differences inabundance between animals infected by M. agalactiae and an-imals from a CA-free flock. Table 3 reports identities, RSC
values, SpC values, and P values for the 68 statistically signif-icant differentially expressed proteins (statistically significantproteins are plotted according to RSC in Fig. S3 in the supple-mental material, and the complete list of statistical results isreported in Table S4 in the supplemental material). A classi-fication based on cellular localization and biological function
FIG. 1. SDS-PAGE of sheep MFGPs and Western blotting results obtained for the immunodominant M. agalactiae antigen P48. (Left) Totalprotein profiles of MFGPs extracted from sheep culturally positive for M. agalactiae (lanes 1 and 2), from culturally negative sheep from the sameflock (lanes 3 and 4), and from sheep belonging to a CA-negative flock (lanes 5 and 6). Total M. agalactiae proteins were loaded in lane 7 as acontrol. (Right) Western immunoblotting with antibodies directed against the immunodominant M. agalactiae lipoprotein rP48. M, molecularweight markers.
was then performed by DAVID on all differential MFGPs,using the normalized protein abundance values to allow forcomparison among localization and functional classes. Asshown in Fig. 4, marked changes were observed both in termsof cellular localization (Fig. 4A) and in terms of biologicalfunction (Fig. 4B). In particular, membrane proteins de-creased, while secreted, mitochondrial, and lysosomal proteinsincreased significantly in positive samples. In terms of biolog-
ical function, membrane and vesicular trafficking proteins de-creased, while enzymes and proteins with immune functionsincreased in positive samples.
The GeLC-MS/MS approach was then applied to the C�/WB� sample (i.e., the third sample analyzed by 2-D DIGE) inorder to evaluate the extent of its protein expression variationand to relate it to truly positive and truly negative samples.Interestingly, the MFGP expression profile for this sheep
FIG. 2. 2-D DIGE of MFGPs from M. agalactiae-infected and uninfected sheep. (Top) Overlay image of MFGPs extracted from representativeinfected (blue) and uninfected (green) milk samples (samples A and D, respectively). Spots indicate proteins with statistically significant differencesin amount among all samples examined. Identities of differentially represented proteins are reported in Table 1. (Bottom) Single-channel imagesof MFGPs extracted from culturally positive sheep milk samples (A and B), from a milk sample with culture negativity and Western immuno-blotting positivity for M. agalactiae (C), and from sheep milk samples from a CA-free flock (D to F).
VOL. 79, 2011 SHEEP MFG PROTEOMICS IN M. AGALACTIAE INFECTION 3837
showed alterations that followed a pattern similar to that forMFGPs of culturally positive sheep, although with a lowerintensity (see Fig. S5 in the supplemental material). Notably,however, for the C�/WB� sheep, several proteins involved inhost defense, immune function, and inflammation, especiallyS100A9, cathelicidin, and lactoferrin, reached high RSC values,as shown in Fig. 5. This might indicate that in such subjects, theexpression levels of proteins involved in lactation and in themechanisms governing milk fat globule secretion are alteredonly slightly, but the inflammation and host defense mecha-nisms are activated and clearly detectable at the proteomiclevel.
In order to investigate the presence of Mycoplasma proteins,the Mycoplasma taxonomy was also searched. As a result, usingthe same stringency parameters used for the mammalian tax-onomy, 15 protein identities were statistically supported by thesoftware. However, a univocal attribution could not be madefor many of these identifications, due to sequence homology
between the sheep and mycoplasma peptides detected. Onlytwo proteins showed sequence differences enabling univocalattribution to mycoplasmas: lactate dehydrogenase, a promi-nent mycoplasma immunogen, and AvgC, a variable surfaceprotein involved in immunological escape. The missed identi-fication by GeLC-MS/MS of relevant M. agalactiae proteins isnot surprising and may be due to several factors dependent onthe sensitivity of the technique and on the proteomic approach,such as identity of the peptide sequences found by MS/MS forbacterial and host homologues, which impairs species attribu-tion, as well as colocalization of bacterial proteins of the samemolecular weight among highly abundant proteins during elec-trophoresis, as might be the case for P48 and butyrophilin,which hampers identification of the less abundant protein spe-cies upon analysis by GeLC-MS/MS due to instrument sensi-tivity constraints.
Identification and pathway analysis of MFGPs showingchanges in abundance upon infection. All proteins showingstatistically significant differences in the protein profiles ofpositive and negative MFG samples and their respective foldchange values were subjected to pathway analysis using IPAsoftware, with the aim of elucidating the main molecular in-teractions and biological connections and representing them bynetworks. Since the IPA database builds on the literature gen-erated for humans and rodents, the UniProt codes for sheepproteins were replaced with the UniProt codes for the closesthuman protein equivalents for the purpose of this analysis.
When all statistically significant differentially abundant pro-teins were subjected to pathway analysis, the network scoringthe highest significance value was cellular movement, hemato-logical system development and function, and immune celltrafficking (score of 64). The diseases and disorders showingthe strongest statistically significant associations with the dif-ferentially expressed proteins were respiratory disease (P val-ues of 1.97E�10 to 3.84E�03), infectious disease (P values of7.55E�10 to 5.20E�03), and the inflammatory response (Pvalues of 1.01E�08 to 7.71E�03), consistent with the patho-gen under examination. The most statistically significant mo-lecular and cellular functions being altered were cellular move-ment (P values of 9.74E�11 to 7.71E�03), cell death (P valuesof 3.04E�10 to 7.71E�03), and free radical scavenging (Pvalues of 1.50E�07 to 7.18E�03) (the complete analysis sum-mary is reported in Table S6 in the supplemental material). Inorder to highlight the different events taking place in infectedMFGs, a pathway analysis was then performed by separatelyexamining proteins significantly increased or decreased inabundance above the 1.5-fold change threshold. Increased pro-teins were associated mostly with the inflammatory response (Pvalues of 1.56E�10 to 2.06E�02), infectious disease (P valuesof 2.18E�08 to 2.06E�02), and respiratory disease (P valuesof 2.18E�08 to 9.27E�03), while decreased proteins wereimplicated mainly in lipid metabolism (P values of 6.18E�07 to2.38E�02), molecular transport (P values of 6.18E�07 to2.38E�02), and small-molecule biochemistry (P values of6.18E�07 to 2.38E�02). Figure 6 depicts the networks withthe highest scores for all differentially abundant proteins (Fig.6A) and for increased (Fig. 6B) and decreased (Fig. 6C) pro-teins. (The complete analysis summaries for increased anddecreased proteins are reported in Tables S7 and S8 in thesupplemental material).
FIG. 3. Statistical analysis of 2-D DIGE results. A score plot (up-per diagram) and heat map (lower diagram) obtained upon compari-son of MFGP samples from M. agalactiae-infected sheep (A, B, and C)with samples from CA-free animals (D, E, and F) are shown. In theheat map, each colored cell represents the protein abundance value fora single sample. Increasingly positive values are indicated by reds ofincreasing intensity, and increasingly negative values are indicated bygreens of increasing intensity. Cells with a value of 0 are colored black.
VOL. 79, 2011 SHEEP MFG PROTEOMICS IN M. AGALACTIAE INFECTION 3839
Western immunoblotting validation of findings for differen-tially abundant MFGPs. Since cytoskeletal and host defenseproteins were among the most intensely increased MFGPs, asindicated also by the GO and IPA analyses, these were evalu-ated by WB of a larger number of samples (n � 21) with thepurpose of validating proteomic results as well as investigatingtheir suitability as indicators of M. agalactiae colonization andhost response. Commercial antibodies against actin, tubulin,S100A9, cathelicidin, and S100A11 were evaluated for reactiv-ity with MFG protein extracts, and the results were comparedto those for PCR, culture, and positivity for the M. agalactiaelipoprotein P48. The results are summarized in Table 1 andillustrated in Fig. 7.
MFGP samples positive for M. agalactiae, by either cul-ture or PCR, displayed strong signals for the host defenseproteins tested by WB, especially S100A9, whose signal wasclearly detectable in all 16 PCR-positive samples tested withthis antibody. Antibodies against cathelicidin and S100A11produced clear positive bands for only a subset of M. aga-
lactiae-positive samples. On the other hand, negative sam-ples did not display any detectable signal for any of the hostdefense proteins revealed to be expressed upon M. agalac-tiae infection, while the same showed a slight positivity forcortical cytoskeleton proteins. Therefore, validation with alarger number of samples from different CA-affected flocksconfirmed the observations obtained with the proteomic anddata analysis approaches and further highlighted the signif-icant and specific increase of the host defense proteinS100A9 in M. agalactiae-positive samples. However, itshould be considered that the apparently smaller amounts ofcathelicidin and S100A11 than of S100A9 seen by Westernimmunoblotting, although consistent with the observationsmade by 2-D DIGE and GeLC-MS/MS, might be due todifferences in specificity of the commercially available anti-bodies used in this work, which are not specific for sheepproteins. In this respect, this should be considered only avalidation of their increased abundance in affected animalsfrom different flocks, while quantitative conclusions should
FIG. 4. Normalized spectral abundance of MFGPs in M. agalactiae-infected (white) and uninfected (black) sheep. Proteins were categorizedby DAVID according to cellular localization (A) and function (B). Asterisks indicate statistically significant differences between the two groupsaccording to a two-tailed t test with a 95% confidence level.
VOL. 79, 2011 SHEEP MFG PROTEOMICS IN M. AGALACTIAE INFECTION 3841
be drawn only by means of quantitative proteomic ap-proaches or alternative quantitative assays.
DISCUSSION
This report presents a comprehensive evaluation of pro-teomic changes occurring in MFGs of sheep infected by M.agalactiae, providing new information on the in vivo eventstaking place in the lactating mammary epithelium during abacterial infection. Specifically, a combined approach based on2-D DIGE, GeLC-MS/MS, and pathway analysis was appliedto study the protein profile of MFGs produced by lactatingepithelial cells during a natural M. agalactiae infection. Takentogether, the different approaches converged on consistent re-sults. Increased levels of proteins involved in inflammation andimmune defense (such as antimicrobial proteins and peptides),in folding (such as HSPs), and in the cortical cytoskeleton weredetected in infected sheep MFGs. Increased amounts of pro-teins involved in oxidative stress, such as the mitochondrialsuperoxide dismutase, were also observed, consistent with thefindings of other researchers upon transcriptome studies of thebovine mammary gland infected with Escherichia coli (26). Onthe other hand, there was an evident and significant decreasein abundance of proteins devoted to the physiological func-tions of MFGs, i.e., lipid synthesis, transport, and secretion. InMFGs from infected animals, S100A9 (calgranulin B) was theprotein showing the most upregulation, with a striking averagepositive/negative ratio of 61.46 upon 2-D DIGE analysis, fol-lowed by S100A11 (calgizzarin) and cathelicidin-1, proteinsinvolved in inflammation/host defense, as also confirmed byGeLC-MS/MS and immunoblotting. With all the techniquesapplied in this study, S100 proteins and cathelicidins werenever detected in MFGs from healthy, M. agalactiae-freesheep, demonstrating their specific and significant increase
during infection. Nevertheless, an abundance of these proteins,together with other host defense proteins, was clearly detectedin MFG extracts obtained from sheep infected by M. agalactiaebut negative by milk culture, i.e., with lower bacterial loads; infact, S100A9, S100A11, cathelicidin, and the myeloid antimi-crobial peptide were overrepresented, while proteins involvedin milk fat secretion/metabolism were slightly underrepre-sented.
S100 proteins and cathelicidins are known to possess proin-flammatory functions and direct antimicrobial effects (5, 23),and their upregulation is increasingly reported for microbialinfections. Recent proteomic and transcriptomic studies re-ported a significant increase of S100 proteins and cathelicidinsin milk during natural and experimental bovine mastitis (19,26), although their cellular source was not investigated. How-ever, an increase in mRNAs for host defense, cytoskeletal, andheat shock proteins was recently demonstrated for the bovinemammary epithelial cell line MAC-T infected with differentbacterial pathogens, suggesting an in vivo role in their produc-tion for mammary epithelial cells. High levels of S100 proteinsand cathelicidins have been reported for several epithelial tis-sues, mostly associated with both acute and chronic inflamma-tory and infectious conditions, such as genitourinary infections,arthritis, psoriasis, and degenerative disorders (15, 29, 35, 50–52), further supporting the hypothesis that these high levelsmight also occur in the mammary epithelium. Yet the contri-bution of phagocytic cells cannot be ruled out completely, sincephagocytic cells with engulfed MFGs, possibly opsonizing bac-teria or bacterial proteins, might hypothetically contaminatethe fat ring preparation due to their lower density, despiterepeated and extensive washing and centrifugation.
Interesting insights into the role played by the proinflamma-tory S100 proteins in the response of epithelial cells to infec-
FIG. 5. Comparison of RSC values of selected MFGPs. Bars indicate the protein levels in C�/WB� sheep (black) and C�/WB� sheep (gray)compared to MFGP levels in uninfected sheep.
tion were recently provided by a study on Candida infection ofmouse vaginal epithelia (51). The authors of that study de-tected an increase of both S100 mRNAs and proteins in in-fected epithelial cells, leading to recruitment of polymorpho-nuclear neutrophils (PMNs) in the vagina, with the extentdepending on the amount of proinflammatory mediators pro-duced by epithelial cells. Similarly, since the mammary gland isexposed to a variety of microbial pathogens in the environ-ment, a first line of defense is built up by lactocytes. The results
obtained here indicate that exploitation of MFGs as surrogatesof the lactocyte cytoplasm has the potential to provide clues tothe extent of the mammary gland response to infection in vivoand to elucidate the role played by lactocytes in the establish-ment of acute or chronic infections by M. agalactiae.
Indications of the pathways involved in mastitis were pro-vided by the IPA analysis performed on all MFGPs undergoingsignificant changes in abundance upon infection by M. agalac-tiae. In particular, in the category of diseases and functions,
FIG. 6. Results of Ingenuity pathway analysis. The highest-scoring networks for all differentially represented proteins (A) and for proteinsoverrepresented (B) and underrepresented (C) in infected sheep are illustrated. (A and B) Results for the highest-scoring network, i.e., cellularmovement, hematological system development and function, and immune cell trafficking, are illustrated. (C) Results for the highest-scoringnetwork, i.e., lipid metabolism, molecular transport, and small-molecule biochemistry, are illustrated. Red, overrepresented proteins; green,underrepresented proteins; white, proteins indicated by IPA as significantly associated with the reported network; continuous line, directrelationship; dotted line, indirect relationship. Color intensity represents the extent of differential protein abundance.
VOL. 79, 2011 SHEEP MFG PROTEOMICS IN M. AGALACTIAE INFECTION 3843
IPA highlighted an increase in proteins associated with respi-ratory disease, infectious disease, and the inflammatory re-sponse. In evaluating the pathway analysis data, it must betaken into account that IPA builds on the literature findings forhumans and rodents; therefore, the results generated uponIPA analysis of our protein expression database are biased byfindings on human infections. Notably, clinically significanthuman mycoplasma infections occur mainly in the airways(e.g., M. pneumoniae) and the genitourinary tract (e.g., M.hominis and M. genitalium). This could explain in part why IPAanalysis of our data generated a significant score for respira-tory disease in the category of diseases and disorders and mightunderline similarities in the host responses elicited by myco-plasmas upon epithelial tissue infection in different hosts, aswell as pointing out common traits in the innate immune re-sponse mechanisms elicited in different secretory epithelia. It isintriguing that the respiratory epithelium (20, 32) and manyother secretory epithelia are increasingly being reported toproduce exosomes involved in innate defense and immuno-modulation (33, 49), such as biliary duct cells (47), intestinalepithelial cells (48), and most interestingly, the human mam-mary gland (3). Since sheep MFGs present many of the phys-icochemical attributes and proteomic signatures of exosomes(25, 31), functions associated with these secretory vesiclesmight likely be observed in MFGs during infection of themammary gland.
Finally, important insights into disease diagnosis and controlcan be drawn from this study. Currently, the cultural and im-munological tools available for detection of M. agalactiae pro-vide a rapid diagnosis of disease but may not be very sensitivefor use with chronically affected herds and flocks. The limita-tions of microbial culture, which currently remains the mostwidespread method for assessing the presence of M. agalactiaein milk, clearly emerged in this work. In fact, despite positivityby milk PCR and evident macroscopic alterations which im-paired separation of the fat ring, several samples were negativefor M. agalactiae culture (Table 1). This is a known problem,likely due to unreported administration of antimicrobialagents, to acidification of milk causing a reduced viability ofmycoplasmas, or to the massive presence of other bacteria that
may hinder the growth of mycoplasmas in selective agar plates(46). Another reason for culture negativity can be a very lowbacterial load, as might have occurred in this work for severalmilk samples which were negative for mycoplasma culture, hada normal appearance, and formed an apparently normal fatring but were positive by milk PCR and by Western immuno-blotting for P48 and S100A9. This occurrence might be ofparticular relevance, as it is the typical condition encounteredduring more insidious chronic, subclinical infections or at thefirst stages of infection. Here we observed that some hostproteins are strongly overrepresented upon infection of themammary gland and behave as diagnostic antigens with a levelof sensitivity comparable to that of milk PCR. Future studiesmight enable the exploitation of these proteins as sensitive andeasily detectable markers of mammary infection and inflam-mation. Moreover, should their increase also be demonstratedupon infection by other microbial pathogens, as suggested bystudies of cow mastitis, these proteins may also possess futurepotential as pathogen-independent indicators of sheep mam-mary infection, providing a simple, sensitive, and comprehen-sive tool for rapidly detecting chronic infections in asymptom-atic carrier animals and for preventing their spread to thewhole flock or herd.
In summary, this is the first report detailing the proteomicchanges occurring in MFGs of sheep naturally infected by abacterial pathogen. The results reported here open the way toelucidation of the molecular events taking place in the infectedmammary epithelium in vivo, offer valuable insights for under-standing the changes induced by M. agalactiae in its naturalhost, and possess significant potential for the development oftools enabling diagnosis and control of chronic, subclinicalinfections of the mammary gland in sheep.
ACKNOWLEDGMENTS
We thank Roberto Tonelli for his help with the statistical processingof data.
This work was supported by funding from the Regione Autonomadella Sardegna—Progetto Cluster Proteomica.
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Editor: A. J. Baumler
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