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1、RESEARCH ARTICLECandida albicans induces mucosal bacterialdysbiosis that promotes invasive infectionMartinna BertoliniID1,Amit Ranjan1,Angela Thompson1,Patricia I.DiazID1,Takanori Sobue1,Kendra MaasID2,Anna Dongari-Bagtzoglou1*1 Departmentof Oral Health Sciences,University of Connecticut Health Cent
2、er,Farmington,Connecticut,United States of America,2 Microbial Analysis,Resources,and Services Core,University of Connecticut,Storrs,Connecticut,United States of America*adongariuchc.eduAbstractInfectious complications are a common cause of morbidity and mortality in cancer patientsundergoing chemot
3、herapy due to increased risk of oral and gastrointestinal candidiasis,candidemia and septicemia.Interactions between C.albicans and endogenous mucosalbacteria are important in understanding the mechanisms of invasive infection.We publisheda mouse intravenous chemotherapy model that recapitulates ora
4、l and intestinal mucositis,and myelosuppression in patients receiving 5-fluorouracil.We used this model to study theinfluence of C.albicans on the mucosal bacterial microbiome and compared global commu-nity changes in the oral and intestinal mucosa of the same mice.We validated 16S rRNAgene sequenci
5、ng data by qPCR,in situ hybridization and culture approaches.Mice receiv-ing both 5Fu and C.albicans had an endogenous bacterial overgrowth on the oral but notthe small intestinal mucosa.C.albicans infection was associated with loss of mucosal bac-terial diversity in both sites with indigenous Steno
6、trophomonas,Alphaproteobacteria andEnterococcus species dominating the small intestinal,and Enterococcus species dominat-ing the oral mucosa.Both immunosuppression and Candida infection contributed tochanges in the oral microbiota.Enterococci isolated from mice with oropharyngeal candidia-sis were i
7、mplicated in degrading the epithelial junction protein E-cadherin and increasing thepermeability of the oral epithelial barrier in vitro.Importantly,depletion of these organismswith antibiotics in vivo attenuated oral mucosal E-cadherin degradation and C.albicans inva-sion without affecting fungal b
8、urdens,indicating that bacterial community changes representovert dysbiosis.Our studies demonstrate a complex interaction between C.albicans,theresident mucosal bacterial microbiota and the host environment in pathogenesis.We shedsignificant new light on the role of C.albicans in shaping resident ba
9、cterial communities anddriving mucosal dysbiosis.Author summaryIn cancer patients receiving high dose chemotherapy mucosal candidiasis is common andcan lead to invasive,systemic fungal infection with mortality ranging 2530%.We showedPLOS Pathogens|https:/doi.org/10.1371/journal.ppat.1007717April 22,
10、20191/30a1111111111a1111111111a1111111111a1111111111a1111111111OPEN ACCESSCitation:BertoliniM,RanjanA,ThompsonA,DiazPI,SobueT,MaasK,etal.(2019)Candidaalbicansinducesmucosalbacterialdysbiosisthatpromotesinvasiveinfection.PLoSPathog15(4):e1007717.https:/doi.org/10.1371/journal.ppat.1007717Editor:Mairi
11、C.Noverr,LouisianaStateUniversityHealthSciencesCenterNewOrleans,UNITEDSTATESReceived:December3,2018Accepted:March19,2019Published:April22,2019Copyright:2019Bertolinietal.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,a
12、ndreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.DataAvailabilityStatement:All16sV4DNAsequencingrawdatahavebeendepositedinNCBI,SRAaccession:PRJNA531284.Dataareaccessibleatthefollowinglink:http:/www.ncbi.nlm.nih.gov/bioproject/531284.Funding:WorkwassupportedbyNIHgrantsDE013986a
13、ndDE023632toADB.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.that in chemotherapy-immunosuppressed mice C.albicans induces a dysbiotic switch thatfavors growth of enterococci on mucosal surfaces,particularly on the oral mucosa.Over-growth
14、of these organisms led to enhanced oral mucosal barrier breach by C.albicans.Like C.albicans,Enterococcus species are a major concern in critical care patients due totheir involvement in sepsis and resistance to multiple antibiotics.There is mounting evi-dence that C.albicans and Enterococci co-exis
15、t in human disease samples.Our discoveriesprovide the first experimental evidence of pathogenic synergy between these microorgan-isms in the context of dysbiosis.IntroductionOropharyngeal(OPC)and gastrointestinal candidiasis are common infections in patientson high dose cancer chemotherapy,mostly at
16、tributed to Candida albicans.In these popula-tions prevalence rates of OPC range between 2540%1.Cytotoxic chemotherapy alsocauses an inflammatory form of oral and gastrointestinal injury known as mucositis 2.Mucosal injury combined with the myelosuppressive effects of chemotherapy,promotebacterial a
17、nd fungal translocation through mucosal barriers leading to bloodstream infec-tions,a major cause of morbidity and mortality in this patient population 3,4.There issome evidence that the oral and intestinal bacterial microbiota in humans may be alteredby cytotoxic chemotherapy,although the effects o
18、n the fungal microbiota are less clear5,6.In a healthy host,unperturbed resident commensal bacterial communities play animportant role in limiting C.albicans colonization in mucosal sites 7.However,when themicrobial equilibrium is changed by immunosuppression certain bacterial species mayovergrow an
19、d form mutualistic relationships with C.albicans.This in turn may lead to awell-coordinated dysbiosis which amplifies mucosal damage.We recently revealed mutual-istic relationships between C.albicans and commensal oral streptococci in a mouse modelof OPC 8,9,10,11.However,these studies were performe
20、d with bacteria that are not partof the indigenous mouse microbiota.A role for indigenous bacterial community-mediateddysbiosis in fungal pathogenesis has never been examined.Furthermore,the effects of C.albicans in modulating commensal bacterial community composition have only been stud-ied in the
21、mouse gastric and intestinal mucosa.In these studies C.albicans was shown tofavor growth of endogenous enterococci post-antibiotics treatment 12,13,14.Studiesfocusing on the interplay between C.albicans and resident oral mucosa bacteria in healthand disease are nonexistent.Chemotherapy-associated mu
22、cosal candidiasis studies use high doses of chemotherapeuticagents and focus exclusively on the development of gastrointestinal or disseminated candidia-sis.The vast majority of these studies also used antibiotics aimed at increasing gastrointestinalfungal burdens 15,16,17,18.We recently developed a
23、 mouse model of low dose intravenous5Fu administration that recapitulates the histopathologic changes associated with cancer che-motherapy-induced mucositis 19.Using this model we tested the hypothesis that mucosalinjury combined with peripheral neutropenia induced by 5Fu,are sufficient for the deve
24、lop-ment of oropharyngeal and intestinal candidiasis in mice with unperturbed resident bacterialmicrobiota.For the first time we also examined the influence of 5Fu and candidiasis on themucosal bacterial microbiome and the reciprocal effect of the resident microbiota on C.albi-cans virulence.Candida
25、 albicans induces mucosal bacterial dysbiosisPLOS Pathogens|https:/doi.org/10.1371/journal.ppat.1007717April 22,20192/30Results5Fu predisposed mice to oropharyngeal and intestinal candidiasis,increased oral bacterial burdens and disseminated infectionIn the first series of experiments we tested whet
26、her intravenous,low dose 5Fu administrationincreases the susceptibility of mice to oral and intestinal candidiasis.Fig 1A shows that micereceiving four doses of 5Fu(50 mg/kg every other day)developed tongue papillary atrophy,epi-thelial desquamation and erosion,consistent with early stages of mucosi
27、tis 19,20.While thistreatment caused gradual depletion of mature neutrophils in the bone marrow and blood,asteady infiltrate of CD11b+/LyG+cells was found in the tongue mucosa reflecting local inflam-mation secondary to mucosal injury(S1A,S1B and S1C Fig).Mice receiving 5Fu+C.albicansdeveloped thick
28、 white biofilms covering the posterior tongue surface,associated with epithelialulcerations(Fig 1A).In these mice C.albicans fungal burdens increased significantly over timein all mucosal surfaces(Fig 1B).This group also lost significantly more weight than mice treatedwith 5Fu alone(Fig 1C).We also
29、noted almost complete absence of neutrophils in the tonguesof this group(S1A,S1B and S1C Fig).This was not due to absence of neutrophil activating cyto-kines since KC and IL-6 were significantly increased in infected tissues(S1D Fig).In mice treated with 5Fu and infected with C.albicans,tongue biofi
30、lms were composed byC.albicans and indigenous bacteria(Fig 1A,lower panel).The PBS control group receiving C.albicans showed complete absence of biofilms and pathology and an early increase in tongueneutrophils consistent with other reports(Figs 1A and S1B)21.No bacterial biofilms wereseen on the su
31、rface of 5Fu-only or C.albicans-only treated mice using a pan-eubacterial FISHprobe(Fig 1A,lower panel).The presence of endogenous bacteria in biofilms with C.albicans prompted a closer exami-nation of the resident bacterial microbiota.We first compared viable(CFU)and total(qPCR)bacterial biomass ac
32、ross all experimental groups at the end of the infection period.As seen inFig 1D there was a significant increase in the viable and total bacterial biomass in mice treatedwith 5Fu and infected with C.albicans,compared to all other groups.We also noted a signifi-cant increase in endogenous bacteria w
33、ith 5Fu treatment alone,compared to untreated controland C.albicans alone groups.However,since the increase in bacterial biomass with 5Fu didnot lead to the development of biofilms(Fig 1A),this suggested that in the absence of C.albi-cans bacteria could not organize in biofilm community structures o
34、n the tongue surface.A positive correlation was found in fungal and bacterial loads on the same tongues at theend of the experimental period(S2A Fig),suggesting that fungal burdens increased in concertwith endogenous bacterial burdens.To confirm this we examined time-dependent changes inthe viable a
35、nd total bacterial biomass on the tongue mucosa of mice in this group.S2B Figshows a gradual increase in bacterial biomass over time,confirming that endogenous bacteriaincreased as C.albicans burdens rose(Fig 1B).Collectively these data show that C.albicansinfection in 5Fu-treated mice promotes bact
36、erial overgrowth on the oral mucosa and that 5Futreatment contributes to this effect.In contrast,mucosa-associated bacterial loads did not change significantly after 8 days ofinfection in the jejunum of mice with candidiasis(S2C Fig),suggesting that the effects of C.albicans infection on commensal b
37、acteria are mucosal site-specific.Strain SC5314 colonizedthe jejunum of PBS control mice in low numbers(not shown)and this was associated with asignificant reduction in resident bacterial CFUs(S2D Fig).A smaller decrease in the viablebacterial biomass was also noted in mice receiving both 5Fu and C.
38、albicans.These results sug-gested that growth of C.albicans in this mucosal site displaces endogenous bacterialcommunities.Candida albicans induces mucosal bacterial dysbiosisPLOS Pathogens|https:/doi.org/10.1371/journal.ppat.1007717April 22,20193/30Fig 1.Effect of 5Fu administration on Candida albi
39、cans and endogenous bacterial burdens.A:Mucosal biofilm forming on tongues excised 8 days post-treatment.Thegroup receiving 5Fu(50mg/kg,IV,every 48 hours)+C.albicans SC5314(5Fu+Ca)had a thick mucosal biofilm covering the posterior tongue surface.Middle panelincludes H&E-stains showing reduced epithe
40、lial thickness,papillary atrophy,desquamation and erosion in the 5Fu group and deep epithelial ulcerations in the 5Fu+Cagroup.Lower panel shows immunofluorescence combined with fluorescence in situ hybridization(immuno-FISH)to simultaneously visualize C.albicans and bacterialcommensals in tongue bio
41、films.C.albicans stained with a polyclonal anti-Candida antibody(green),commensal bacteria stained with EUB338-Alexa 546 probe(red)andcell nuclei counterstained with Hoechst 33258(blue).B:Recovery of C.albicans from tongues,esophagus and small intestines in control untreated mice,and in micereceivin
42、g 5Fu every 48h with C.albicans SC5314 added daily in the drinking water.Shown are colony-forming unit(CFU)counts from organs harvested at baseline(uninfected),then 2,6 and 8 days after the first 5Fu injection.Tissues were weighed,homogenized,serially diluted and plated for counts on Sabouraud Dextr
43、ose Agarcontaining chloramphenicol.Similar counts of C.albicans were obtained in CHROMagar Candida media with no other species identified(not shown).CFU assaysCandida albicans induces mucosal bacterial dysbiosisPLOS Pathogens|https:/doi.org/10.1371/journal.ppat.1007717April 22,20194/30Cytotoxic chem
44、otherapy elevates the risk for bloodstream infections 22,we thus askedwhether C.albicans disseminated in distant organs.We found a time-dependent increase infungal burdens in kidneys and livers,accompanied by increased bacterial burdens in the sameorgans(Fig 1E).Collectively these data suggest that
45、5Fu creates favorable conditions for C.albicans and endogenous bacterial growth in the upper and lower alimentary tract mucosaeand systemic dissemination of bacteria and fungi.Importantly,mice receiving both 5Fu and C.albicans had an endogenous bacterial overgrowth in the oral mucosa that exceeded 5
46、Fu treat-ment alone.In 5Fu-treated mice OPC was associated with loss of bacterial diversity andindigenous enterococcal bloomThe increase in oral bacterial burdens observed in mice with OPC raised the possibility ofglobal mucosa-associated bacterial microbiota changes.We thus performed high throughpu
47、tsequencing of the V4 hypervariable region of the 16S rRNA gene in DNA extracted from ton-gues.We also analyzed the bacterial microbiome of the jejunum in the same mice,for compar-ison.To explore differences in bacterial community composition within each treatment andcontrol groups we first analyzed
48、 alpha diversity as reflected by Shannon index(Fig 2A)andrichness estimates(S3A Fig).Compared to the untreated group the 5Fu group was not signifi-cantly different in community diversity when either the tongue or jejunum were examined.However,inoculation with C.albicans alone induced a decrease in b
49、acterial diversity in theoral mucosa,whereas diversity in the jejunum increased(Fig 2A),consistent with other reports23.This illustrates that daily inoculation with this strain had an impact on oral biodiversityeven though colonization was below the sensitivity limit of the CFU assay.A dramatic drop
50、 inbacterial diversity was noted in the tongues of mice receiving C.albicans and 5Fu(Fig 2A),consistent with the reduction in the number of species(OTUs)observed(S3A Fig).A signifi-cant reduction in the average number of species was also observed in the jejunum of the samemice(S3A Fig).To further ex