deletion of braun lipoprotein gene (lpp) attenuates yersinia pestis kimd27 strain role of lpp in modulating host immune response

1、Deletion of Braun lipoprotein gene (lpp) attenuates Yersinia pestis KIMD27 strain Role of Lpp in modulating host immune responseDeletion of Braun lipoprotein gene (lpp) attenuates Yersinia pestis KIM/D27 strain:Role of Lpp in modulating host immune response, NF- k B activation and cell deathTie Liu1

2、 , Stacy L. Agar 1 , Jian Sha, Ashok K. Chopra *Department of Microbiology & Immunology, Medical Research Building, University of Texas Medical Branch, Galveston, TX 77555-1070, USAa r t i c l e i n f oArticle history:Received 2 May 2021Received in revised form25 August 2021Accepted 1 Septem

3、ber 2021Available online 6 September 2021Keywords:Yersinia pestisBraun lipoproteinMouse model of infectionNF- k BApoptosisHost immune responsea b s t r a c tThe pathogenic species of yersiniae potently blocks immune responses in host cells by using the type IIIsecretion apparatus and its effector pr

4、oteins. In this study, we characterized potential mechanismsassociated with the Braun lipoprotein (Lpp) that contributed to a further attenuation of a pigmentationlocus-minus Yersinia pestis KIM/D27 mutant strain and its ability to generate immune responses in mice.The lpp gene encodes one of the ma

5、jor outer membrane lipoproteins that is involved in inflammatoryresponses and septic shock. We found that sera and splenocytes from D lpp mutant-immunized mice,when transferred to na? ¨ve animals, provided protection to the latter against challenge with a lethal doseof the Y. pestis parental st

6、rain. Further, the D lpp mutant promoted ex vivo a significantly higher inter-leukin (IL)-2 and interferon-gamma production from Tcells of immunized mice, when compared to thosefrom animals infected with the sub-lethal dose of the parental Y. pestis KIM/D27 strain. Likewise, murineprimary macrophage

7、s infected with the mutant, when compared to those infected with the parentalstrain in vitro, produced significantly higher IL-12 levels. Importantly, increased nuclear factor-kappa Bactivation and decreased apoptosis were noted in splenocytes and primary macrophages of mice chal-lenged with the D l

8、pp mutant, when compared to those in animals infected with the parental Y. pestisKIM/D27 strain. Finally, significantly higher levels of antibodies specific for the parental Y. pestis antigenswere developed in mice first immunized with the D lpp mutant and then challenged with the parentalstrain, co

9、mpared to the antibody levels in animals that were immunized and then infected with theparental KIM/D27 strain. To our knowledge, this is the first report of a mechanistic basis for attenuationand immunological responses associated with deletion of the lpp gene from the Y. pestis KIM/D27 strain.? 20

10、21 Elsevier Ltd. All rights reserved.1. IntroductionThe genus Yersinia includes three human pathogenic species,Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotubercu-losis. Y. pestis is the causative agent of bubonic and pneumonicplague, both of which remain serious public health threa

11、ts in someregions of the world, accounting for the deaths of approximately200 million people throughout recorded history 1. Y. pestis isgenerally transmitted to humans via the bite of an infected rodentflea and is endemic to Africa, India, and the southwestern UnitedStates 24. Because plague is high

12、ly infectious and can readilyspread by aerosolization, it poses a bioterrorism threat 2,5.Yersiniae type III secretion system (T3SS) expression is inducedby contact with host cells in a temperature-dependent fashionresulting in secretion and/or translocation of Yersinia outermembrane proteins (Yops)

13、, including the low calcium response Vantigen (LcrV) 68. The T3SS and its effectors Yops are encoded ona 70-kb plasmid present in all of the pathogenic species of yersiniae1. In addition, Y. pestis synthesizes an anti-phagocytic capsule, thegenes for which are encoded on a 110-kb plasmid 1. Yersinia

14、e thatcan successfully translocate Yops into the host cytosol are capableof modulating the hosts response to infection, thereby increasingtheir own survival 9.Lipoproteins are outer membrane modified proteins with anN-terminal diacyl cysteine and are present in many bacterialpathogens 10,11. Some of

15、 these lipoproteins are important viru-lence factors during the transmission, colonization and persistenceof bacterial pathogens in the host 1215. For example, Braunlipoprotein (Lpp), specific for gram-negative bacteria of Enter-obacteriaceae family, has been shown to trigger the innate immunerespon

16、se and to induce apoptosis of the host cell through the toll-like receptor (TLR)-2 16.* Corresponding author. Tel.: t1 409 747 0578; fax: t1 409 747 6869.E-mail address: (A.K. Chopra).1Contributed equally to this manuscript.Contents lists available at ScienceDirectMicrobial Pathogene

17、sisjournal homepage: elsevier /locate/micpath0882-4010/$ see front matter ? 2021 Elsevier Ltd. All rights reserved.doi:10.1016/j.micpath.2021.09.002Microbial Pathogenesis 48 (2021) 4252The human innate immune response to bacterial infectionbegins with the recognition by TLRs of pathogen-associatedmo

18、lecular patterns, such as lipopolysaccharide (LPS), lipoprotein,and flagella 17. TLR stimulation initiates a signaling cascade thatresults in NF- k B activation and the downstream production ofproinflammatory cytokines 17. The different signaling pathwaysconverge at the IKK complex, which consists o

19、f two catalyticsubunits, IKK a and IKK b , and the regulatory subunit, NF- k Bessential modulator (NEMO)/IKK g . The activated IKK complexphosphorylates the NF- k B inhibitory I k B proteins that sequester thetranscription factor NF- k B in the cytoplasm of unstimulated cells.I k B phosphorylation l

20、eads to NF- k B liberation, therebyallowing thetranslocation of NF- k B tothe nucleus and activation of transcriptionof several inflammation-associated genes 18,19. A recent study onYersinia indicated that YopJ (YopP in Y. enterocolitica), a T3SSeffector, interrupts the crucial proinflammatory respo

21、nse early ininfection by inhibiting NF- k B activation 20. NF- k B also plays animportant role in regulating the expression of anti-apoptoticproteins (e.g., c-IAP-1/2, AI, Bcl-2 and Bcl-X L ) and the cell-cycleregulator, cyclin D1, which are necessary in cellular survival andproliferation, respectiv

22、ely 21,22.Apoptosis is a type of programmed cell death that is morpho-logically defined by cellular and nuclear shrinkage, chromatincondensation, DNA fragmentation, and the formation of apoptoticbodies. It is a mechanism typically used during fetal developmentand in adult cell maintenance by elimina

23、ting potentially harmfulcells without causing an inflammatory response. Several bacteria,such as the Yersinia species, Shigella flexneri, and Helicobacter pylori,exploit or interact with the apoptotic pathway(s) to enhance theinfection process 2325. Yersinia triggers programmed cell deathin cultured

24、 macrophages 26, and YopJ/P is necessary for inducingtheir apoptosis 2729.In this study, we immunologically characterized the D lppmutant of a pigmentation locus (pgm)-minus strain of Y. pestis KIM,which was designated as KIM/D27. It has been shown that thedeletion of the 102-kb pgm locus from the c

25、hromosome of Y. pestisKIM attenuates its virulence 1. In our recent study, we demon-strated that the D lpp mutant of Y. pestis KIM/D27 was more atten-uated than was the parental strain in a mouse model of infection,and animals immunized with this mutant were protected againstinfection with the virul

26、ent strain CO92 of Y. pestis via the inhala-tional route 30. We now provided evidence that Y. pestis KIM/D27D lpp mutant induced higher levels of cytokine production from Tcells and macrophages, evoked increased NF- k B activation, andresulted in decreased apoptosis of macrophages and splenocytes of

27、mice, when these parameters were compared to findings inanimals infected with the parental Y. pestis KIM/D27 strain. This isa first study in which a mechanistic basis of attenuation caused bydeletion of the lpp gene from Y. pestis KIM/D27 strain was exploredin in vitro and in vivo models and the rol

28、e of Lpp in modulating thehost immune response studied.2. Results2.1. Protection of mice immunized with the D lpp mutant of Y. pestisKIM/D27 against various lethal challenge doses of the parentalstrainWe immunized Swiss-Webster mice via the intraperitoneal (i.p.)route with the D lpp mutant at a non-

29、lethal dose of 5?10 6 colonyforming units (cfu). These mice were then challenged i.p. with theparental Y. pestis KIM/D27 strain at various doses 30 days afterimmunization with the D lpp mutant. We opted for the i.p route ofchallenge, as bacteria could be seen in all of the organs of miceinfected via

30、 the intranasal/aerosol route with Y. pestis CO92 and thisi.p. route mimics development of bubonic plague 30,31. In theimmunized group of mice that received 1?10 8 cfu of parental Y.pestis, 80% of them died, while 60% of the animals in the immunizedgroup given 5?10 7 cfu of parental bacteria died. I

31、n the group ofimmunized animals thatreceived1?10 7 cfu ofparental bacteria,wenoted an 80% survival, while no mice died in the immunized groupthat was challenged with 5?10 6 cfu of parental bacteria (data notshown).Theseobservationsindicatedthatthe D lppmutantofY.pestisKIM/D27 retained its immunogeni

32、city to provide dose-dependentprotection against subsequent challenge with the parental strain.2.2. Protective effect of sera and splenocytes from the D lpp mutant-immunized mice against challenge with parental Y. pestis KIM/D27strain in na? ¨ve miceIn the next set of experiments, mice were i.p

33、. immunized witha sub-lethal dose (5?10 5 cfu) of either the parental Y. pestis KIM/D27 strain or its D lpp mutant. Sera from these mice were collectedafter 14 days, and an aliquot (100 m l) was injected intravenously(i.v.) into na? ¨ve mice. After 24 h, these animals were infected i.p.with 5?1

34、0 6 cfu of parental bacteria, and monitored for mortality.As shown in Fig. 1a, there was a 100% survival following parentalDays Post Infection30 28 26 24 22 20 8 7 6 5 4 3 2 1 0Percent Survival00 20 40 60 81000 1 x 5 , l a t n e r a P ( l o r t n o c6) u f ccontrol splenocytess e t y c o n e l p s l

35、 a t n e r a P lpp splenocytes*Days Post Infection30 28 26 24 22 20 8 7 6 5 4 3 2 1 0Percent Survival00 20 40 60 80 0 10 1 x 5 , l a t n e r a P ( l o r t n o c6) u f cm u r e s l o r t n o cm u r e s l a t n e r a Pp p l m u r e s *abFig.1. Protective effect of sera (a) and splenocytes (b) from par

36、ental- and D lpp mutant-infected mice. Five mice were infected i.p. with 5 ?10 5 cfu of either parental Yersiniapestis KIM/D27 or its D lpp mutant. After 14 days, sera or splenocytes were collectedand injected i.v. into na? ¨ve mice (10 per group). Control sera were collected from na? ¨vem

37、ice. After 2448 h, these animals were rechallenged with 5 ?10 6 cfu of parental Y.pestis KIM/D27 strain. The p values (p< 0.05) were in comparison to the groups having100% survival and shown by asterisks.T. Liu et al. / Microbial Pathogenesis 48 (2021) 4252 43strain challenge of the na? ¨

38、;ve animals passively administered withsera from mice that were either immunized with parental Y. pestisKIM/D27 or with its D lpp mutant. On the contrary, only 40% of theanimals survived in a group that received an injection of controlserum from uninfected mice. Additionally and expectedly, 60% ofth

39、e animals given phosphate-buffered saline (PBS) instead of anysera, but challenged with the parental bacterium (control group),died within 24 days, which matched with our bacterial doseresponse data in mice (data not shown).We also removed the spleens and isolated splenocytes frommice infected for 1

40、4 days with 5?10 5 cfu of either parental Y. pestisKIM/D27 or its D lpp mutant. These splenocytes (5?10 5 ) were theninjected via the i.v. route into na? ¨ve mice. After 48 h, these animalswere challenged with the parental Y. pestis KIM/D27 strain at5?10 6 cfu. As was previously demonstrated in

41、 the serum experi-ment, 100% of the na? ¨ve animals that received splenocytes frommice initially immunized with either the parental Y. pestis KIM/D27or its D lpp mutant were protected (Fig. 1b). However, only 50% ofthe mice immunized with splenocytes of uninfected animals(control splenocytes) s

42、urvived, and 60% of the mice that were notimmunized, but infected with parental bacteria (control), suc-cumbed to infection (Fig. 1b).2.3. Cytokine production by primary macrophages and from T cellsof animals infected with parental Y. pestis KIM/D27 strain or its D lppmutantCytokines, such as interf

43、eron (IFN)- g and interleukin (IL)-12p40, are important components of the protective responses againstY. enterocolitica infections in mice 32,33. Therefore, to elucidatewhether deletion of the lpp gene from the parental Y. pestis KIM/D27 strain would augment production of these cytokines andenhance

44、the host immune response, we infected mice with5?10 5 cfu of the parental bacteria or its D lpp mutant via the i.p.route. After 5 days, the spleens of these mice were removed, andthe splenocytes isolated and enriched for T cells on nylon woolcolumns. These T cells were cultured for 48 h before we me

45、asuredthe levels of IFN- g and IL-2 in the culture supernatants. As shown inFig. 2a and b, significantly higher levels of IFN- g (16,000 pg/ml) andIL-2 (7800 pg/ml) were detected in T cells from mice infected withthe D lpp mutant compared to those levels in uninfected controls(2000 and 2800 pg/ml, r

46、espectively, for IFN- g and IL-2). Similarly,we noted higher levels of IFN- g from Tcells of D lpp mutant-infectedmice compared to those found in animals infected with theparental Y. pestis KIM/D27 strain (9000 pg/ml) (Fig. 2a). However,IL-2 levels were similar in the supernatants of T cells infecte

47、d withthe parental Y. pestis strain and the control (Fig. 2b).Next, primary macrophages were isolated from na? ¨ve mice asdescribed (Section 4.4) and infected with either the parental Y.pestis KIM/D27 strain or its D lpp mutant. The IL-12 p40 levels weresignificantly higher in macrophages infec

48、ted with the D lpp mutant(9000 pg/ml) when compared to the levels in both the uninfectedcontrols (1000 pg/ml) and the parental Y. pestis-infected macro-phages (6000 pg/ml) (Fig. 2c). The level of IL-12 from macrophagesinfected with the parental Y. pestis KIM/D27 was also significantlyhigher compared

49、 to that in uninfected host cells.2.4. Y. pestis-induced host cell death in vitro and in vivoPrimary macrophages (3?10 6 ) from na? ¨ve mice were infectedwith 3?10 6 cfu (multiplicity of infection MOI of 1) of eitherparental Y. pestis KIM/D27 strain or its D lpp mutant for 24 h inDulbeccos Modi

50、fied Eagle Medium (DMEM) using 6-well plates,which were incubated at 37? C and 5% CO 2 . Macrophages were thenstained with 0.4% trypan blue solution and visualized under lightmicroscopy for cell viability (Section 4.4). Viable cells remainedunstained, while blue-stained cells represented a loss of c

51、ellularviability. After culturing macrophages with the parental Y. pestisKIM/D27 strain, we noted an increase in cell death (72%), either as00 0 0 20 0 0 40 0 0 60 0 0 80 0 0 0 10 0 0 2 10 0 0 4 10 0 0 6 10 0 0 8 10 0 0 0 2l o r t n o C p p l l a t n e r a PConcentration of IFN- (pg/ml)* * *00 0 0 2

52、0 0 0 40 0 0 60 0 0 80 0 0 0 1l o r t n o C p p l l a t n e r a PConcentration of IL-2 (pg/ml)* * *5 0 . 0 > p* * *00 0 0 20 0 0 40 0 0 60 0 0 80 0 0 0 10 0 0 2 1l o r t n o C p p l l a t n e r a PConcentration of IL-12 p40 (pg/ml)* * *abcFig. 2. Cytokine production by T cells from animals in

53、fected either with the parentalYersinia pestis KIM/D27 or its D lpp mutant. Mice were infected via the i.p. route with5 ?10 5 cfu of either parental Y. pestis KIM/D27 or its D lpp mutant. After 5 days, thespleens from these mice and na? ¨ve animals were removed, and the Tcells were isolatedand

54、cultured for an additional 48 h. The supernatants of the cultured T cells werecollected, and the IFN- g (a) and IL-2 (b) levels were measured. Macrophages fromna? ¨ve mice were cultured with either parental Y. pestis KIM/D27 or its D lpp mutant atan MOI of 0.5. After 24 h, the macrophages were

55、cultured for an additional 48 h. Thesupernatants from cultured macrophages were collected and assayed for the presenceof IL-12 p40 (c). The actual p values comparing each of the groups are shown*p <0.01; *p< 0.001.T. Liu et al. / Microbial Pathogenesis 48 (2021) 4252 44a result of apop

56、tosis or necrosis (Fig. 3a, Panel C), while only 31% ofthe host cells cultured with the D lpp mutant were apparentlyapoptotic/necrotic (Fig. 3a, Panel B). A minimal number (7%) ofmacrophages died in the PBS control group (Fig. 3a, Panel A). Weexamined a minimum of 25 fields and the mean numbers ofap

57、optotic/necrotic cells (plotted as percentage) with standarddeviations using parental Y. pestis and its D lpp mutant are shown inFig. 3a, Panel D.Likewise, splenocytes were isolated from the spleens of miceinfected with 5?10 5 cfu of either the parental Y. pestis KIM/D27strain or its D lpp mutant fo

58、r 5 days and were then stained withtrypan blue solution to assess cell viability. All of the splenocytesfrom uninfected control mice, shown in Fig. 3b, Panel A, appearednormal (unstained) after trypan blue staining. Splenocytes (8090%) from mice infected with the parental Y. pestis KIM/D27 strain(Pa

59、nel C) were significantly enlarged and exhibited blebbing,a hallmark of apoptosis, when we compared them with those hostcells infected with the D lpp mutant (Panel B) in which no apparentcell death was noted. We examined a minimum of 25 fields and themean numbers of apoptotic/necrotic splenocytes (plotted aspercentage)with standard deviations using parental Y. pestis and itsD lpp mutant are shown in Fig. 3b, Panel D.To confirm Y. pestis-induced apoptosis/necrosis, we cultured exvivo splenocytes and thymocytes of mice with Y. pestis KIM/D27strain or its D lpp