route of injection affects the impact of inlb...

Research ArticleRoute of Injection Affects the Impact ofInlB Internalin Domain Variants on Severity ofListeria monocytogenes Infection in Mice

Konstantin A Sobyanin Elena V Sysolyatina Yaroslava M ChalenkoEgor V Kalinin and Svetlana A Ermolaeva

Gamaleya Research Center of Epidemiology and Microbiology Moscow 123098 Russia

Correspondence should be addressed to Svetlana A Ermolaeva drermolaevamailru

Received 8 September 2017 Accepted 29 November 2017 Published 28 December 2017

Academic Editor Jozef Anne

Copyright copy 2017 Konstantin A Sobyanin et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The facultative intracellular pathogen Listeria monocytogenes causes a severe food-borne infection in humans and animals Lmonocytogenes invasion factor InlB interacts with the tyrosine kinase c-Met via the N-terminal internalin domain Previouslydistinct variants of the InlB internalin domain (idInlB) have been described in L monocytogenes field isolates Three variantswere used to restore full-length InlB expression in the L monocytogenes strain EGDeΔinlB Obtained isogenic L monocytogenesstrains were tested in the invasion assay and intravenous intraperitoneal and intragastric models of infection in mice All idInlBswere functional restored InlB activity as an invasion factor and improved invasion of the parental strain EGDeΔinlB into humankidney HEK23 cells Meanwhile distinct idInlBs provided different mortality rates and bacterial loads in internal organs Whenrecombinant strains were compared the variant designated idInlB14 decreased severity of disease caused by intravenous andintraperitoneal bacterial administration whereas this variant improved intestine colonization and stimulated intragastric infectionObtained results demonstrated that naturally occurring idInlBs differed in their impact on severity of L monocytogenes infectionin mice in dependence on the infection route

1 Introduction

The Gram-positive bacterium Listeria monocytogenes is acausative agent of food-borne infection listeriosis in humansandmany domestic andwild animals [1 2] Listeriosis is a rarebut severe infection with lethality reaching 30 in humans[3 4] The most serious clinical manifestations of listeriosisare CNS disorders and fetus impairment in pregnant females

The intestine is a port of bacterial entry in the courseof food-borne listeriosis [1 5] L monocytogenes crosses theintestinal barrier and colonizes the lamina propria fromwhere it spreads to internal organs [5] Systemic listeriosisincludes colonization of the liver and spleen If the infection isnot controlled at this stage a secondary bacteremia developsand bacteria cross cerebral andor maternal-fetal barriers toinfect the brain and fetus [1 2 5 6]

L monocytogenes is a facultative intracellular pathogenthat infectsmacrophages and awide range of nonprofessional

phagocytes [7] Internalin (InlA) and InlB are major factorsthat provide active invasion in nonprofessional phagocytes[7 8] InlA and InlB belong to the internalin family Inter-nalin family proteins include an internalin domain whichconsists of a central leucine rich repeat- (LRR-) domainflanked by amino-terminal N-cap and carboxy-terminalimmunoglobulin-like (Ig-like) domains [9] The internalindomain is directly involved in specific protein-protein inter-actions with mammalian cell surface receptors InlB inter-nalin domain specifically interacts with the eukaryotic recep-tor c-MetHGFR (Hepatocyte Growth Factor Receptor) [10]

Mouse models are of key importance for studies of Lmonocytogenes virulence LD50 and bacterial counts in theliver and spleen are major ways used for assessment of Lmonocytogenes strain virulence [11 12] Intravenous (iv) andintraperitoneal (ip) infection routes usually require a smallerinfective dose and are used more often in L monocytogenesresearch than intragastric (ig) or oral routes [13ndash15] When

HindawiBioMed Research InternationalVolume 2017 Article ID 2101575 8 pageshttpsdoiorg10115520172101575

2 BioMed Research International

Table 1 Strains and plasmids used in the work

Speciesstrain Characteristics ReferenceL monocytogenes

EGDe1 Serovar 12a type strain [27]EGDeΔinlB1 EGDe derivative with inlB gene deletion [28]

VIMHA004 Serovar 4b ST22 clinical isolate obtained from thestillborn carried idInlB13 [21]

VIMHA015 Serovar 4b ST12 Clinical isolate obtained from thestillborn carried idInlB93 [21]

VIMHA034 Serovar 12a ST3142 Clinical isolate obtained from thestillborn carried idInlB143 [21]

EGDeΔinlBInlB1 EGDeΔinlB supplemented with the pInlB1 plasmid [25]EGDeΔinlBInlB9 EGDeΔinlB supplemented with the pInlB9 plasmid [25]EGDeΔinlBInlB14 EGDeΔinlB supplemented with the pInlB14 plasmid [25]

E coli

JM109endA1 glnV44 thi-1 relA1 gyrA96 recA1mcrB+Δ(lac-proAB) e14-[F1015840 traD36

proAB+lacIqlacZΔM15] hsdR17(rK-mK+)Promega

BL21 (DE3) FndashompT gal dcm lon hsdSB(rB-mB-) 120582(DE3 [lacIlacUV5-T7 gene 1 ind1 sam7 nin5])

NewEnglandBioLabs

PlasmidspTgem-easy Cloning vector PromegapTRKH2 Shuttle-vector [29]pInlAB pTRKH2PromoterinlABgwdInlB

4 [25]pInlB1 pInlABidInlB1 [25]pInlB9 pInlABidInlB9 [25]pInlB14 pInlABidInlB14 [25]

1The strain was generously provided by Professor J A Vazquez-Boland University of Edinburgh 2Sequence tyes (STs) are provided according to the MLSTprotocol by Ragon et al 2008 3idInlB is an internalin domain of InlB 4gwdInlB is InlB GW-domain

several strains were compared their relative virulence wasdependent on the route of infection Certain strains highlyvirulent when injected ip or ip are less virulent whenapplied orally or ig and vice versa [16 17]

L monocytogenes oral route infection in mice is depen-dent on InlB which accelerates listerial invasion into M cellsof ileal Peyerrsquos patches (PPs) [18 19] Previously we andothers described isolation of L monocytogenes from internalorgans of wild small rodents belonging to the Murinaesubfamily which is closely related to Mus musculus Thesedata suggest that mouse intestine barrier crossing is effectiveenough for systemic infection to be established in the naturalenvironment [20 21] Among 15 InlB variants found in Lmonocytogenes strains only two variants were revealed inL monocytogenes isolated from wild small rodents [21 22]Existence of specific InlB variants was demonstrated for Lmonocytogenes strains characterized by reduced virulence[23] or associated with cardiovascular disease [24]

To evaluate an impact of naturally occurring InlB variantson L monocytogenes virulence we used a set of previouslydescribed isogenic strains that carried distinct InlB internalindomains (idInlBs) [25] Virulence of the recombinant strainswas compared in mouse models Distinct idInlBs provideddifferent mortality rates and bacterial loads in internal

organs and the difference was dependent on the route ofinfection

2 Materials and Methods

21 Bacterial Strains andGrowthConditions Bacterial strainsused in the work are listed in Table 1 L monocytogeneswere routinely cultivated in the Brain Heart Infusion (BHIBD USA) medium To get a virulence regulon induction Lmonocytogenes was grown in BHI supplemented with 02activated charcoal (Merck) [26] E coli used in cloning andexpression procedures were cultivated in the LB medium(Sigma-Aldrich) All bacteria were grown at 37∘C with agita-tion at 180 rpm Antibiotics were added up to the followingconcentrations erythromycin up to 10 and 300 120583gmlminus1 forL monocytogenes and E coli respectively ampicillin up to100 120583gmlminus1 kanamycin up to 200 120583gmlminus1 All antibioticswere purchased from Sigma-Aldrich To prepare a culture forinfection bacteria were grown up to mid-exponential phasewashed with PBS aliquoted and frozen in the presence of10 glycerin The concentration was determined by platingserial dilutions from the frozen culture the day before theexperiment Bacteria were thawed immediately before the

BioMed Research International 3

experiment and resuspended in PBS up to the requiredconcentration

22 Mammalian Cells and Cultivation Conditions Humanembryonic kidney HEK-293 cells (the public collection ofGamaleya Center httpvirologygamaleyaorgindexphpoption=com contentampview=articleampid=103kollektsiya-kle-tochnykh-kulturampcatid=91ampItemid=645) were used Cellswere grown in the DMEM medium supplemented with 10FBS (fetal bovine serum) in the 5 CO

2atmosphere In

some experiments FBS was decreased up to 2

23 Recombinant Strains Recombinant strain constructionwas described previously [25] Shortly the idInlB encodingDNA fragments obtained on lysates of L monocytogenesstrains VIMHA004 VIMHA015 and VIMHA034 (Table 1)with primers InlB3 51015840-CAAGCGGGATCCATCACCGTG-CCAACGCC and InlB4 51015840-TATCCGGGATCCTGCTTC-TACTTTTG were cloned into the derivative of the pTRKH2vector [29] carrying the inlB gene lacking the internalindomain encoding part The obtained plasmids encoded thefull-length InlB with different idInlBs The plasmids wereincorporated into the L monocytogenes EGDeΔinlB strain byelectroporation

24 Cell Invasion Assay Bacteria were prepared from themid-exponential culture as it was described above Theldquogentamycin assayrdquo was performed as described previously[25] Briefly bacteria resuspended inDMEMwere addedwithMOI 100 1 (bacteria to cell) to cells incubated 1 h at 37∘Cin 5 CO

2atmosphere and washed with PBS and then the

fresh medium supplemented with 100120583gmlminus1 gentamycin(Fluka) was added In 1 h cells were washed and lysedwith 1 Triton X-100 Serial dilutions of cell lysates wereplated to count bacterial coloniesThe invasion efficiency wasdetermined as the ratio of intracellular bacteria to the numberof applied bacteria

25 Mice Experiments on animals were conducted in accor-dance with the Russian Federation National Standard (GOSTR52379-2005) directives of Ministry of Health of RussianFederation (number 753n from 26082010 number 774Yfrom 31082010) and with the approval of the BiomedicalEthics Committee of Gamaleya Research Center of Epidemi-ology and Microbiology (number 93 from 22102015) Ani-mals were sacrificed using ether inhalation Female BALBcmice of 16ndash18 g purchased from the nursery Stolbovaya(Moscow region Russia) were kept at our breeding facilitiesin specific-pathogen-free conditions and used in all experi-ments

26 Experimental Infection in Mice For intravenous infec-tion mice were prewarmed with air heater and injected intothe tail vein with bacteria suspended in 100120583l PBS Thedoses are indicated in the text For intraperitoneal infectionmice were injected intraperitoneally with bacteria suspendedin 200120583l PBS For intragastric infection mice starved for12 h were anesthetized by intraperitoneal injection of sodiumpentobarbital (40120583gg) and infected with bacteria suspended

in 200120583l PBS by using the needle with the diameter 06mmand the length 25mm The tip of the needle was cut andpolished to smoothness to avoid stomach injury

27 Mortality Rates and Bacterial Loads in Organs To deter-mine mortality rates groups of 20 animals were infected ivwith doses of bacteria higher or near LD50 The experimentwas stopped as soon as 50 lethality was observed in at leastone of the groups and all remained animals were euthanized

Groups of 5 animals were used to determine bacterialloads in infected organs Animals were euthanized with etherat time points specified in the text The liver and the spleenwere aseptically harvested and homogenized in 1 and 02mlsterile PBS respectively The intestine was washed out withsterile PBS visible Peyerrsquos patcheswere separated andhomog-enized in 5ml of sterile PBS Small intestine was separatelyhomogenized in 5ml of sterile PBS Tenfold serial dilutionsof organ homogenates were plated on BHI agar supplementedwith antibiotics if required Plates were incubated at 37∘C for24 h The detection limit of this procedure was 10 10 and 102colony forming units (CFU) per liver spleen and intestinerespectively

28 Statistics All experiments were performed using dupli-cate samples Results of repeated experiments were averagedThe one-tailed 119905-test was used for assessment of statisticalsignificance

3 Results

31 idInlB Variants Differentially Supported iv L monocyto-genes Infection Firstly we evaluated an impact of idInlB vari-ants on the final outcome of intravenous (iv) infection Tocomparemortality rates 2times 106 CFUof parental EGDeΔinlBwild-type EGDe or each recombinant strain was injectedinto the tail vein of female BALBc mice This bacterial loadwas near LD50 for the parental strain EGDeΔinlB (LD50 asymp1 times 106 in our experimental settings) and much higher thanLD50 for the wild-type strain EGDe (LD50 asymp 15 times 104) The50 mortality was reached in 72 h after infection with bothEGDeΔinlB and the wild-type strain EGDe (Figure 1) thatwas in line with the high infection dose Mortality caused byall but one recombinant strains had similar rates Howeverbacteria carrying idInlB14 caused noticeably lower mortalityrates with only 5 mortality during the first three days(Figure 1(a))

To understand whether the strain EGDeΔinlBInlB14was able to multiply within the host we followed bacterialloads in the liver and spleen of mice infected with 104bacteria of this strain A noticeable drop in bacterial loadswas observed 24 hpi in comparison with the dose appliedfor infection only (35 plusmn 23) times 103 CFU per mouse werefound in the liver and (53 plusmn 18) times 102 CFU per mouse in thespleen (Figure 1(b)) However after the initial drop bacterialloads increased logarithmically suggesting that the strain iscompetent to multiply within the host

To check whether the same drop happens with all otherstrains and at high infection doses and to establish at what


Perc

enta

ge o

f sur

vivo

rs

20 40 60 800hpi

0

20

40

60

80

100

idInlB9idInlB1EGDeΔinlB

EGDeidInlB14

(a)

LiverSpleen

20 40 60 800hpi

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

ＦＩＡ５

per m

ouse

(b)EG

De

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

ＦＩＡ10

CFU

per

live

r

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

ＦＩＡ10

CFU

per

sple

en

(c)

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09

ＦＩＡ10

CFU

per

sple

en

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09

ＦＩＡ10

CFU

per

live

r

(d)

Figure 1 Impact of idInlB variants on severity of intravenous infection in BALBc mice (a) Mortality caused by parental EGDeΔinlB wild-type EGDe and recombinant EGDeΔinlBInlB1 (idInlB1) EGDeΔinlBInlB9 (idInlB9) and EGDeΔinlBInlB14 (idInlB14) strains Groupsof 20 mice were infected iv with 2 times 106 CFU as described in Materials and Methods (b) Proliferation of EGDeΔinlBInlB14 in the liver andspleen of mice iv infected with 1 times 104 CFU Average and SD for groups of 5 mice are shown (c) and (d) idInlB-dependent drop of bacterialloads in the liver and spleen 6 (c) and 24 (d) hpi BALBc mice were iv infected with 1 times 106 CFU The data and average of two independentexperiments performed on groups of 5 mice are shown


time point it takes place we compared bacterial loads inthe liver and spleen for all strains 6 and 24 h after ivinfection with 106 bacteria By 6 hpi the number of wild-type EGDe bacteria was higher than EGDeΔinlB in boththe liver (492 times 105 versus 27 times 105 CFU per mouse resp119901 lt 005) and the spleen (297 times 104 versus 24 times 104 CFU permouse) The recombinant strain EGDeΔinlBinlB9 behavedsimilarly to the parental with average values of 247 times 105and 220 times 104 CFU per liver and spleen respectively Thestrain EGDeΔinlBinlB1 behaved more closely to the wild-type strain (457 times 105 and 24 times 104 CFU per liver andspleen resp) The strain EGDeΔinlBinlB14 demonstratedthe lowest loads in both the liver and spleen (625 times 104 and125 times 103 CFU per liver and spleen resp 119901 lt 005)

By 24 hpi EGDe andEGDeΔinlB accumulated in the liverto 108 times 108 and 647 times 107 CFU per mouse respectivelywhile EGDeΔinlBInlB1 and EGDeΔinlBInlB9 accumulatedto slightly lower values (232 times 107 and 122 times 107 CFU permouse resp 119901 lt 005 Figure 1(d)) The difference betweenEGDeΔinlB and EGDeΔinlBInlB14 reached 2 log 10 (119901 lt001) In the spleen all recombinant strains accumulated tovalues noticeably lower than control bacteria (119901 lt 005)The difference between EGDeΔinlBInlB14 and the parentalstrain in the spleen reached 276 log 10 (742 times 107 and 127 times105 CFU per mouse for EGDeΔinlB and EGDeΔinlBInlB14resp 119901 lt 001)

Taken together obtained results demonstrated that (i)InlB hyperproduction rather worsened than improved bacte-rial accumulation in the first 24 h this effect was especiallynoticeable in the spleen 24 hpi and (ii) InlB internalindomain variants differed in their ability to support ivinfection with the idInlB14 demonstrating the worst results

32 idInlBs Differed in Their Ability to Support ip InfectionIn contrast with iv infection all recombinant strains hadimproved bacterial loads in comparison with the parentalstrain EGDeΔinlB when 106 bacteria were injected intraperi-toneally (ip) and bacterial loads in the liver and spleen weredetected 72 hpi (Figure 2 119901 lt 005) However pairwisestrain comparison demonstrated that the EGDeΔinlBInlB9supplied noticeably higher infection levels than the variantsEGDeΔinlBInlB1 and EGDeΔinlBInlB14 The effect wasmore pronounced in the liver recombinant strain countswere higher comparatively to the parental (9306 plusmn 2342)-fold for EGDeΔinlBInlB9 and (64 plusmn 18)- and (26 plusmn 6)-foldfor EGDeΔinlBInlB1 and EGDeΔinlBInlB14 respectivelyLoads of the wild-type EGDe strain were (4954 plusmn 1582)-higher thanEGDeΔinlBThe same tendencywas observed forcolonization of the spleen the counts of EGDeΔinlBInlB9and wild-type bacteria exceeded the counts of the parentalstrain (577 plusmn 97)- and (586 plusmn 70)-fold respectively whilethe counts of EGDeΔinlBInlB1 and EGDeΔinlBInlB14exceeded parental (63 plusmn 8)- and (60 plusmn 11)-fold only

33 idInlB9 and idInlB14 Differentially Support Intestine Col-onization Previously we have demonstrated that the idInlBsdiffered in their ability to support intragastric (ig) infectionproviding different bacterial loads in the liver but not in the

Liver Spleen100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

idInlB1idInlB9idInlB14

EGDe

Figure 2 Impact of id InlB variants on severity of intraperi-toneal infection in BALBc mice Results are shown as a rela-tive increase of bacterial loads in the liver and spleen for therecombinant and wild-type strains comparatively with the strainEGDeΔinlB which lacked the inlB gene The strains are designatedas follows EGDeΔinlBInlB1 idInlB1 EGDeΔinlBInlB9 idInlB9EGDeΔinlBInlB14 idInlB14 and EGDe wild-type strain EGDe

spleen [25] The idInlB14 gave rise to the highest loads in theliver 3 days after ig inoculation In contrast idInlB9 whichas we demonstrated here provided high bacterial loads afterip injection was low efficient in ig route infection [25]

The internal organ penetration after ig infection is notan instantaneous event as it happens after iv injection Colo-nization of the intestine was shown to play an important roleupon ig infection [30] To check whether distinct idInlBsdifferentially affect gut colonization EGDeΔinlBInlB9 andEGDeΔinlBInlB14 strains were applied ig in the dose of108 CFU per mouse and the number of bacteria in the villousepithelium and Peyerrsquos patches was counted 24 hpi To countboth intracellular and extracellular bacteria associated withthe intestine the intestine was washed thoroughly but itwas not treated with gentamycin so both intracellular andsurface attached bacteria could be counted Counting ofEGDeΔinlBInlB14 bacteria was about 15 log 10 higher thanEGDeΔinlBInlB9 bacteria in Peyerrsquos patches (Figure 3 119901 lt005) The difference between strains for villous epitheliumwas not so high about 3-fold (not significant)

Taken together obtained results demonstrated that InlBinternalin domain variants differed in their ability to supportinfection in dependence on the route of bacterial injectionunder otherwise equal conditions The idInlB14 was lesseffective when bacteria were injected iv while idInlB9 wasless effective upon ig infection The third variant idInlB1demonstrated medium results in all tests

34 All idInlBs Provided Invasion in Epithelial Cells InlBpromotes L monocytogenes invasion in epithelial cells Tocheck whether idInlBs differ in their ability to supportbacterial invasion in epithelial cells human kidney carci-noma HEK293 cells were infected with recombinant and


idInlB14idInlB90100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06ＦＩＡ10

CFU

per

mou

se

(a)idInlB9 idInlB140

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

ＦＩＡ10

CFU

per

mou

se

(b)

Figure 3 Impact of id InlB variants 9 and 14 on colonization of the intestine Bacterial loads in Peyerrsquos patches (a) and villous epithelium (b)were determined for groups of 5 BALBc mice infected intragastrically with 108 CFU of EGDeΔinlBInlB9 (idInlB9) and EGDeΔinlBInlB14(idInlB14)

EGDΔinlB idInlB9 idInlB14idInlB100001

0001

001

01

1

Inva

sion

effici

ency

Figure 4 Impact of id InlB variants on invasion efficiency in humanHEK293 epithelial cells Cells were infected with MOI 100 and theinvasion efficiency is shown as a ratio of intracellular bacteria to thenumber of bacteria used for infectionThe average and SE of at least3 independent experiments performed in duplicate are shown

control bacteria (MOI 100 1 bacteria cells) Relative inva-sion of EGDeΔinlBInlB9 was 27-fold higher in HEK293than EGDeΔinlBInlB14 (Figure 4) However the differencebetween recombinant strains was minor in comparison withinvasion of the parental strain EGDeΔinlB that was about2 log 10 lower for both cell lines Therefore all idInlBs werefunctional to support invasion in epithelial cells

4 Discussion

Mouse models are of key importance for studies of listeriosisLmonocytogenes strains differ considerably in their virulence[11 12] Moreover an infection route affects severity ofL monocytogenes infection The intragastric (ig) route isusually noticeably less effective than intravenous (iv) andintraperitoneal (ip) routes with LD50 differed by 4ndash6 log 10[30 31] Still other works reported a smaller differencewithin 1-2 log 10 or a better effectiveness of the ig infectionwhen alternative L monocytogenes strains were used [16 17]Isolation of L monocytogenes from the internal organs of

wild small rodents captured in the pristine habitats furthersupports the view that systemic L monocytogenes infectionin mice might take place via the oral route even at low dosesof infection that might be expected in natural ecosystems[20 22 32]

Previously we suggested that the sequence of the invasionfactor InlB might be critical for L monocytogenes spreadingamong small rodents in the pristine environment [21 25]Indeed InlB is a key invasion factor responsible for theintestine barrier crossing in mice [19] Among 15 InlB inter-nalin domain variants described in L monocytogenes isolatedfrom different hosts only two variants were discovered instrains isolated fromwild small rodents [21 25]These are theInlB internalin domain variant 1 designated here the idInlB1which was found in strains belonging to the phylogenetic lin-eage I and the variant idInlB14 found in the lineage II strainsComparison with available data established that idInlB14 wasfound in the widely used type strains 10403 and EGD but notin EGDe which was used in this work as a control and whichcarries the distinct idInlB [21 25 33] Besides listed strainsidInlB14 was described by Temoin et al in a group of strainsthat demonstrated low virulence in iv infected mice [23]Complementation of low virulent strains with the plasmidencoding InlB from the strain EGDe improved virulenceon the iv infection model [23] Our data are in line withthese results demonstrating that idInlB14 caused a decrease inbacterial counts after iv infection (see Figure 2) Meanwhilethe idInlB14 variant effectively supported the intragastricinfection [25] and intestine colonization (see Figure 3)

5 Conclusions

Obtained results demonstrated that naturally occurring InlBinternalin domain variants differed in their impact on theoutcome ofLmonocytogenes infectionThe impact of distinctidInlBs was determined by the route of infection The resultssupported the view that L monocytogenes virulence on themodels of intravenous and intraperitoneal mouse infectionis not fully consistent with virulence on the intragastricroute infection model Particular idInlB variants might beresponsible for this inconsistency


Conflicts of Interest

The authors have no conflicts of interest to declare

Acknowledgments

Theauthors are thankful to Professor T R Klaenhammer andProfessor J A Vazquez-Boland for providing the pTRKH2vector and the L monocytogenes EGDeΔinlB strain respec-tively The work was supported by RSF (Grant 16-15-00091)

References

[1] J A Vazquez-Boland M Kuhn P Berche et al ldquoListeriapathogenesis and molecular virulence determinantsrdquo ClinicalMicrobiology Reviews vol 14 no 3 pp 584ndash640 2001

[2] A Oevermann A Zurbriggen andMVandevelde ldquoRhomben-cephalitis caused by listeria monocytogenes in humans andruminants A zoonosis on the riserdquo Interdisciplinary Perspec-tives on Infectious Diseases vol 2010 Article ID 632513 2010

[3] S Kathariou ldquoListeria monocytogenes virulence and pathogen-icity a food safety perspectiverdquo Journal of Food Protection vol65 no 11 pp 1811ndash1829 2002

[4] C M De Noordhout B Devleesschauwer F J Angulo et alldquoThe global burden of listeriosis a systematic review and meta-analysisrdquoThe Lancet Infectious Diseases vol 14 no 11 pp 1073ndash1082 2014

[5] M Lecuit ldquoUnderstanding how Listeria monocytogenes targetsand crosses host barriersrdquo Clinical Microbiology and Infectionvol 11 no 6 pp 430ndash436 2005

[6] O Disson and M Lecuit ldquoTargeting of the central nervoussystem by Listeria monocytogenesrdquo Virulence vol 3 no 2 pp213ndash221 2012

[7] P Cossart J Pizarro-Cerda and M Lecuit ldquoInvasion of mam-malian cells by Listeria monocytogenes Functional mimicry tosubvert cellular functionsrdquo Trends in Cell Biology vol 13 no 1pp 23ndash31 2003

[8] L Braun S Dramsi P Dehoux H Bierne G Lindahl and PCossart ldquoInIB An invasion protein of Listeria monocytogeneswith a novel type of surface associationrdquo Molecular Microbiol-ogy vol 25 no 2 pp 285ndash294 1997

[9] W-D Schubert G Gobel M Diepholz et al ldquoInternalins fromthe human pathogen Listeria monocytogenes combine threedistinct folds into a contiguous internalin domainrdquo Journal ofMolecular Biology vol 312 no 4 pp 783ndash794 2001

[10] Y Shen M Naujokas M Park and K Ireton ldquoInIB-dependentinternalization of Listeria is mediated by the Met receptortyrosine kinaserdquo Cell vol 103 no 3 pp 501ndash510 2000

[11] K Hoelzer R Pouillot and S Dennis ldquoAnimal models oflisteriosis A comparative review of the current state of the artand lessons learnedrdquo Veterinary Research vol 43 no 1 articleno 18 2012

[12] S E F DrsquoOrazio ldquoAnimal models for oral transmission ofListeria monocytogenesrdquo Frontiers in Cellular and InfectionMicrobiology vol 4 2014

[13] O Disson G Nikitas S Grayo O Dussurget P Cossart andMLecuit ldquoModeling human listeriosis in natural and geneticallyengineered animalsrdquoNature Protocols vol 4 no 6 pp 799ndash8102009

[14] D Cabanes M Lecuit and P Cossart ldquoAnimal models ofListeria infectionrdquo Current Protocols in Microbiology 2008

[15] E N B Ghanem T Myers-Morales and S E F DrsquoOrazio ldquoAmouse model of foodborne Listeria monocytogenes infectionrdquoCurrent Protocols in Microbiology 2013

[16] L Pine G B Malcolm and B D Plikaytis ldquoListeria monocyto-genes intragastric and intraperitoneal approximate 50 lethaldoses for mice are comparable but death occurs earlier byintragastric feedingrdquo Infection and Immunity vol 58 no 9 pp2940ndash2945 1990

[17] A H Barbour A Rampling and C E Hormaeche ldquoCom-parison of the infectivity of isolates of Listeria monocytogenesfollowing intragastric and intravenous inoculation in micerdquoMicrobial Pathogenesis vol 20 no 4 pp 247ndash253 1996

[18] M Pentecost J Kumaran P Ghosh andM R Amieva ldquoListeriamonocytogenes internalin B activates junctional endocytosis toaccelerate intestinal invasionrdquo PLoS Pathogens vol 6 no 5 pp1ndash15 2010

[19] S Chiba T Nagai T Hayashi Y Baba S Nagai and S KoyasuldquoListerial invasion protein internalin B promotes entry into ilealPeyerrsquos patches in vivordquo Microbiology and Immunology vol 55no 2 pp 123ndash129 2011

[20] E Zaytseva S Ermolaeva and G P Somov ldquoLow genetic diver-sity and epidemiological significance of Listeriamonocytogenesisolated from wild animals in the far east of Russiardquo InfectionGenetics and Evolution vol 7 no 6 pp 736ndash742 2007

[21] R Adgamov E Zaytseva J-MThiberge S Brisse and S Ermo-laeva ldquoGenetic Diversity inMicroorganismsrdquo M Caliskan Edpp 235ndash250 InTEchOpen 2012

[22] O L Voronina N N Ryzhova M S Kunda M A KurnaevaA N Semenov and E I Aksenova ldquoDiversity and pathogenicpotential of listeria monocytogenes isolated from environmen-tal sources in the russian federationrdquo International Journal OfModern Engineering Research vol 5 pp 5ndash15 2015

[23] S Temoin S M Roche O Grepinet Y Fardini and P VelgeldquoMultiple point mutations in virulence genes explain the lowvirulence of Listeria monocytogenes field strainsrdquoMicrobiologyvol 154 no 3 pp 939ndash948 2008

[24] F Alonzo III L D Bobo D J Skiest and N E FreitagldquoEvidence for subpopulations of Listeria monocytogenes withenhanced invasion of cardiac cellsrdquo Journal of Medical Microbi-ology vol 60 no 4 pp 423ndash434 2011

[25] K Sobyanin E Sysolyatina M Krivozubov Y Chalenko AKaryagina and S Ermolaeva ldquoNaturally occurring InlB vari-ants that support intragastric Listeria monocytogenes infectionin micerdquo FEMS Microbiology Letters 2017

[26] S Ermolaeva Y Belyi and I Tartakovskii ldquoCharacteristics ofinduction of virulence factor expression by activated charcoalin ListeriamonocytogenesrdquoFEMSMicrobiology Letters vol 174no 1 pp 137ndash141 1999

[27] P Glaser L Frangeul C Buchrieser C Rusniok A Amend andF Baquero ldquoComparative genomics of Listeria speciesrdquo Sciencevol 294 pp 849ndash852 2001

[28] A Kibardin T Karpova T Sapenko J A Vazquez-Boland SKiselev and S Ermolaeva ldquoMammalian peptidoglycan recogni-tion protein TagL inhibits Listeriamonocytogenes invasion intoepithelial cellsrdquoFEMS ImmunologyampMedicalMicrobiology vol46 no 2 pp 284ndash290 2006

[29] D J OrsquoSullivan and T R Klaenhammer ldquoHigh- and low-copy-number Lactococcus shuttle cloning vectors with features forclone screeningrdquo Gene vol 137 no 2 pp 227ndash231 1993

[30] J A Melton-Witt S M Rafelski D A Portnoy and AI Bakardjiev ldquoOral infection with signature-tagged Listeria


monocytogenes reveals organ-specific growth and dissemina-tion routes in guinea pigsrdquo Infection and Immunity vol 80 no2 pp 720ndash732 2012

[31] M Lecuit S Vandormael-Pournin J Lefort et al ldquoA transgenicmodel for listeriosis Role of internalin in crossing the intestinalbarrierrdquo Science vol 292 no 5522 pp 1722ndash1725 2001

[32] M L Gray and A H Killinger ldquoListeria monocytogenes andlisteric infectionsrdquo Bacteriological Reviews vol 30 no 2 pp309ndash382 1966

[33] C Becavin C Bouchier P Lechat et al ldquoComparison ofwidely used Listeria monocytogenes strains EGD 10403S andEGD-e highlights genomic differences underlying variations inpathogenicityrdquomBio vol 5 no 2 Article ID e00969-14 2014

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Strains and plasmids used in the work

Speciesstrain Characteristics ReferenceL monocytogenes

EGDe1 Serovar 12a type strain [27]EGDeΔinlB1 EGDe derivative with inlB gene deletion [28]

VIMHA004 Serovar 4b ST22 clinical isolate obtained from thestillborn carried idInlB13 [21]

VIMHA015 Serovar 4b ST12 Clinical isolate obtained from thestillborn carried idInlB93 [21]

VIMHA034 Serovar 12a ST3142 Clinical isolate obtained from thestillborn carried idInlB143 [21]

EGDeΔinlBInlB1 EGDeΔinlB supplemented with the pInlB1 plasmid [25]EGDeΔinlBInlB9 EGDeΔinlB supplemented with the pInlB9 plasmid [25]EGDeΔinlBInlB14 EGDeΔinlB supplemented with the pInlB14 plasmid [25]

E coli

JM109endA1 glnV44 thi-1 relA1 gyrA96 recA1mcrB+Δ(lac-proAB) e14-[F1015840 traD36

proAB+lacIqlacZΔM15] hsdR17(rK-mK+)Promega

BL21 (DE3) FndashompT gal dcm lon hsdSB(rB-mB-) 120582(DE3 [lacIlacUV5-T7 gene 1 ind1 sam7 nin5])

NewEnglandBioLabs

PlasmidspTgem-easy Cloning vector PromegapTRKH2 Shuttle-vector [29]pInlAB pTRKH2PromoterinlABgwdInlB

4 [25]pInlB1 pInlABidInlB1 [25]pInlB9 pInlABidInlB9 [25]pInlB14 pInlABidInlB14 [25]

1The strain was generously provided by Professor J A Vazquez-Boland University of Edinburgh 2Sequence tyes (STs) are provided according to the MLSTprotocol by Ragon et al 2008 3idInlB is an internalin domain of InlB 4gwdInlB is InlB GW-domain

several strains were compared their relative virulence wasdependent on the route of infection Certain strains highlyvirulent when injected ip or ip are less virulent whenapplied orally or ig and vice versa [16 17]

L monocytogenes oral route infection in mice is depen-dent on InlB which accelerates listerial invasion into M cellsof ileal Peyerrsquos patches (PPs) [18 19] Previously we andothers described isolation of L monocytogenes from internalorgans of wild small rodents belonging to the Murinaesubfamily which is closely related to Mus musculus Thesedata suggest that mouse intestine barrier crossing is effectiveenough for systemic infection to be established in the naturalenvironment [20 21] Among 15 InlB variants found in Lmonocytogenes strains only two variants were revealed inL monocytogenes isolated from wild small rodents [21 22]Existence of specific InlB variants was demonstrated for Lmonocytogenes strains characterized by reduced virulence[23] or associated with cardiovascular disease [24]

To evaluate an impact of naturally occurring InlB variantson L monocytogenes virulence we used a set of previouslydescribed isogenic strains that carried distinct InlB internalindomains (idInlBs) [25] Virulence of the recombinant strainswas compared in mouse models Distinct idInlBs provideddifferent mortality rates and bacterial loads in internal

organs and the difference was dependent on the route ofinfection

2 Materials and Methods

21 Bacterial Strains andGrowthConditions Bacterial strainsused in the work are listed in Table 1 L monocytogeneswere routinely cultivated in the Brain Heart Infusion (BHIBD USA) medium To get a virulence regulon induction Lmonocytogenes was grown in BHI supplemented with 02activated charcoal (Merck) [26] E coli used in cloning andexpression procedures were cultivated in the LB medium(Sigma-Aldrich) All bacteria were grown at 37∘C with agita-tion at 180 rpm Antibiotics were added up to the followingconcentrations erythromycin up to 10 and 300 120583gmlminus1 forL monocytogenes and E coli respectively ampicillin up to100 120583gmlminus1 kanamycin up to 200 120583gmlminus1 All antibioticswere purchased from Sigma-Aldrich To prepare a culture forinfection bacteria were grown up to mid-exponential phasewashed with PBS aliquoted and frozen in the presence of10 glycerin The concentration was determined by platingserial dilutions from the frozen culture the day before theexperiment Bacteria were thawed immediately before the




2atmosphere In












3 Results





Perc

enta

ge o

f sur

vivo

rs

20 40 60 800hpi

0

20

40

60

80

100


EGDeidInlB14

(a)

LiverSpleen

20 40 60 800hpi

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

ＦＩＡ５

per m

ouse

(b)EG

De

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

ＦＩＡ10

CFU

per

live

r

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

ＦＩＡ10

CFU

per

sple

en

(c)

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09

ＦＩＡ10

CFU

per

sple

en

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09

ＦＩＡ10

CFU

per

live

r

(d)









100E + 01

100E + 02

100E + 03

100E + 04

100E + 05


EGDe








100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06ＦＩＡ10

CFU

per

mou

se


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

ＦＩＡ10

CFU

per

mou

se

(b)



0001

001

01

1

Inva

sion

effici

ency



4 Discussion




5 Conclusions





Acknowledgments


References











































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2atmosphere In












3 Results





Perc

enta

ge o

f sur

vivo

rs

20 40 60 800hpi

0

20

40

60

80

100


EGDeidInlB14

(a)

LiverSpleen

20 40 60 800hpi

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

ＦＩＡ５

per m

ouse

(b)EG

De

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

ＦＩＡ10

CFU

per

live

r

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

ＦＩＡ10

CFU

per

sple

en

(c)

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09

ＦＩＡ10

CFU

per

sple

en

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09

ＦＩＡ10

CFU

per

live

r

(d)









100E + 01

100E + 02

100E + 03

100E + 04

100E + 05


EGDe








100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06ＦＩＡ10

CFU

per

mou

se


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

ＦＩＡ10

CFU

per

mou

se

(b)



0001

001

01

1

Inva

sion

effici

ency



4 Discussion




5 Conclusions





Acknowledgments


References











































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Perc

enta

ge o

f sur

vivo

rs

20 40 60 800hpi

0

20

40

60

80

100


EGDeidInlB14

(a)

LiverSpleen

20 40 60 800hpi

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

ＦＩＡ５

per m

ouse

(b)EG

De

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

ＦＩＡ10

CFU

per

live

r

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

ＦＩＡ10

CFU

per

sple

en

(c)

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09

ＦＩＡ10

CFU

per

sple

en

EGD

e

idIn

lB14

idIn

lB9

idIn

lB1

EGD

eΔin

lB

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

100E + 09

ＦＩＡ10

CFU

per

live

r

(d)









100E + 01

100E + 02

100E + 03

100E + 04

100E + 05


EGDe








100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06ＦＩＡ10

CFU

per

mou

se


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

ＦＩＡ10

CFU

per

mou

se

(b)



0001

001

01

1

Inva

sion

effici

ency



4 Discussion




5 Conclusions





Acknowledgments


References











































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








100E + 01

100E + 02

100E + 03

100E + 04

100E + 05


EGDe








100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06ＦＩＡ10

CFU

per

mou

se


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

ＦＩＡ10

CFU

per

mou

se

(b)



0001

001

01

1

Inva

sion

effici

ency



4 Discussion




5 Conclusions





Acknowledgments


References











































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06ＦＩＡ10

CFU

per

mou

se


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

ＦＩＡ10

CFU

per

mou

se

(b)



0001

001

01

1

Inva

sion

effici

ency



4 Discussion




5 Conclusions





Acknowledgments


References











































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgments


References











































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

route of injection affects the impact of inlb...

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