I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 5 ( 2 0 1 6 ) 1 5 5 –1 6 3

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Expression profile of Rab5, Rab7, tryptophanaspartate-containing coat protein, lepraelipoarabinomannan, and phenolic glycolipid-1on the failure of the phagolysosome processin macrophages of leprosy patients as a viabilitymarker of Mycobacterium lepraeq

http://dx.doi.org/10.1016/j.ijmyco.2016.02.0012212-5531/� 2016 Asian-African Society for Mycobacteriology. Production and hosting by Elsevier Ltd. All rights reserved.

* Corresponding author at: Mayjend Prof. Dr. Moestopo Street, Number 47, Surabaya, Indonesia.E-mail address: citarositasp@yahoo.com (C.R.S. Prakoeswa).

Peer review under responsibility of Asian African Society for Mycobacteriology.

Cita Rosita Sigit Prakoeswa a,*, Ratna Wahyuni b, Iswahyudi b, Dinar Adriaty b,Irawan Yusuf c, Sutjipto d, Indropo Agusni a, Shinzo Izumi b

aDepartment of Dermatology and Venereology, Faculty of Medicine, Universitas Airlangga, Dr. Soetomo Teaching Hospital,

Surabaya, Indonesiab Institute of Tropical Disease, Universitas Airlangga, Surabaya, IndonesiacDepartment of Physiology, Faculty of Medicine, Universitas Hasanuddin, Makassar, IndonesiadDepartment of Biochemistry, Faculty of Medicine, Universitas Airlangga, Dr. Soetomo Teaching Hospital, Surabaya, Indonesia

A R T I C L E I N F O

Article history:

Received 3 January 2016

Received in revised form

2 February 2016

Available online 23 February 2016

Keywords:

Lep-LAM

PGL-1

Rab5

Rab7

TACO

Viable M. leprae

A B S T R A C T

Objective/Background: Phagolysosome process in macrophage of leprosy patients’ is impor-

tant in the early phase of eliminating Mycobacterium leprae invasion. This study was to clar-

ify the involvement of Rab5, Rab7, and trytophan aspartate-containing coat protein (TACO)

from host macrophage and leprae lipoarabinomannan (Lep-LAM) and phenolic glycolipid-1

(PGL-1) from M. leprae cell wall as the reflection of phagolysosome process in relation to 16

subunit ribosomal RNA (16S rRNA) M. leprae as a marker of viability of M. leprae.

Methods: Using a cross sectional design study, skin biopsies were obtained from 47 newly

diagnosed, untreated leprosy at Dr Soetomo Hospital, Surabaya, Indonesia. RNA isolation

and complementary DNA synthesis were performed. Samples were divided into two

groups: 16S rRNA M. leprae- positive and 16S rRNA M. leprae-negative. The expressions of

Rab5, Rab7, TACO, Lep-LAM, and PGL-1 were assessed with an immunohistochemistry

technique.

Result: Using Mann-Whitney U analysis, a significant difference in the expression profile of

Rab5, Rab7, Lep-LAM, and PGL-1 was found (p < .05), but there was no significant difference

of TACO between the two groups (p > .05). Spearman analysis revealed that there was a sig-

nificant correlation between the score of Rab5, Rab7, Lep-LAM, and PGL-1 and the score of

16S rRNA M. leprae (p < .05).

156 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 5 ( 2 0 1 6 ) 1 5 5 –1 6 3

Conclusion: In M. leprae infection, Rab5, Rab7, and Lep-LAM play important roles in the fail-

ure of phagolysosome process via a membrane trafficking pathway, while PGL-1 plays a role

via blocking lysosomal activities. These inventions might be used for the development of

an early diagnostic device in the future.

� 2016 Asian-African Society for Mycobacteriology. Production and hosting by Elsevier Ltd.

All rights reserved.

Introduction

Leprosy is a chronic infection caused by Mycobacterium leprae

infection that may cause damage to skin tissues, nerves, eyes,

or systemic damage. If it continues, it may lead to disabilities

with the impact of reduced quality of life of the individuals

and society [1]. Indonesia is a country with the third highest

number of leprosy patients in the world after India and Brazil,

with the discovery of new cases increasing from year to year

[2]. The most important cause of the high incidence rate and

dominance of the multi-bacillary type of leprosy, which is

potentially infectious in Indonesia, is the high transmission

caused by the difficulty of early detection.

M. leprae cannot be cultured in an artificial medium, so the

study of the bacteria is often difficult. Viability of M. leprae

can be detected by morphological index of acid fast bacilli

using Ziehl Nielsen staining. This examination has low sensi-

tivity and specificity that affect the quality of viability detec-

tion. The detection of 16 subunit ribosomal RNA (16S rRNA)

of M. leprae using the method of reverse transcriptase-

polymerase chain reaction (RT-PCR) can be used as a marker

of viability of M. leprae [3]. The 16S rRNA will be destroyed

after M. leprae dies, thus the study of 16S rRNA using the

RT-PCR method can reflect the viability of M. leprae with high

sensitivity and specificity [4,5]. Although this method is rela-

tively expensive and requires special facilities and infrastruc-

ture, it is relatively easier to perform than radiorespirometric

methods [6–8].

When viable M. leprae in the body of an individual is pre-

sent, theoretically the individual’s phagocytic function has

failed. Phagocytic function failure can occur due to the failure

of the phagolysosomeprocess so thatM. leprae remains persis-

tent. Phagolysosome process failure should be identified early

because there are differences in the profiles of a variety of

compounds derived from M. leprae, as well as from the host

[9,10]. Based on the extrapolation of research that has been

done with other mycobacteria, some proteins are related to

the phagolysosome failure process and the most important

is Rab5 that has been shown to play a role in early endosome

fusion with the phagosome of Mycobacterium tuberculosis and

Mycobacterium bovis bacille Calmette–Guerin (BCG). Rab7 has

been shown to play a role in late endosome fusion with

Mycobacterium marinum phagosome and M. bovis BCG. Trypto-

phan aspartate-containing coat protein (TACO), has been

shown to play a role in resisting the movement of M. bovis

BCG phagolysosome. These three proteins are present in the

host’s macrophage cell membrane [11–13]. In addition, the cell

wall components of M. leprae, the leprae lipoarabinomannan

(Lep-LAM) and phenolic glycolipid-1 (PGL-1), are also thought

to inhibit the phagolysosome by extrapolating from the

research performed on the cell wall of M. tuberculosis [14,15].

However, to date, there remains uncertainty in the expression

profile of Rab5, Rab7, TACO, Lep-LAM, and PGL-1 related to the

effectiveness of the phagolysosome process of M. leprae infec-

tion as reflected by the viability of M. leprae.

Knowing the mechanisms of the host macrophage

response to M. leprae infection, particularly in the phagolyso-

some process, provides an opportunity to solve the problems

of early detection of M. leprae viability. It has been reported

that immunohistochemical examination has good prospects

for early detection of leprosy, especially in endemic countries,

with methods that can be done with simple laboratory equip-

ment [16]. The rationale of immunohistochemical examina-

tion for the detection of M. leprae viability originates from

facts showing that failed phagolysosome process causes M.

leprae persistence.

The purpose of this study was to elaborate Rab5, Rab7, and

TACO (host components) expression profiles and Lep-LAM

and PGL-1 (cell wall components of M. leprae) as a reflection

of the effectiveness processes of the phagolysosome associ-

ated with the presence or absence of 16S rRNA M. leprae as

the marker of M. leprae viability.

Materials and methods

This study was an observational cross-sectional analytic

study to prove that there are differences in the expression

profile of Rab5, Rab7, TACO, Lep-LAM, and PGL-1 in macro-

phages of new leprosy patients based on the presence or

absence of 16S rRNA M. leprae expression, as well as a corre-

lation between 16S rRNA of M. leprae expression with the

expression of Rab5, Rab7, TACO, Lep-LAM, and PGL-1 in new

leprosy patients’ macrophages.

The samples were taken from new leprosy patients over

3 months at the Dermatovenereology Clinic, Dr. Soetomo

Hospital, Surabaya, Indonesia, who met the sample inclusion

criteria. Leprosy diagnosis was made clinically and bacterio-

logically. The sample inclusion criteria were new leprosy

(diagnostic criteria of World Health Organization), aged 15–

40 years, and willing to join the study by signing an informed

consent.

Examination of the 16S rRNA of M. leprae with real time

PCR was performed to obtain more sensitive and specific

results of the examination compared with conventional PCR

[17]. To date, there have been no scientific publications on

the primer used in the examination of the 16S rRNA of M.

leprae with real time PCR. Therefore, the primer and probe

design were done using the Primer Express Software program:

Primer express software version 3.0 Applied Biosystems. At

the P2–P3 (69–239 bp) region there were 50 combinations of

I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 5 ( 2 0 1 6 ) 1 5 5 –1 6 3 157

forward and reserve primers. After primary election by

studying the GC content, melting temperature, and primer

dimer, the forward primers were chosen as follows: P2, F,

P24, and P25 and the reverse primers were P3, R, P34, and

P35 with the order as shown in Fig. 1. The sequences of the

primers are described in Table 1.

After that, forward and reverse primers were combined

according to Applied Biosystems 7300 Real Time PCR Machine

and an optimum primer length between 100 bp and 150 bp, so

the chosen pairs were P2–P3, P2–P34, and P2–P35, and a sensi-

tivity test was done using probes to increase specificity, with

the results as shown in Table 2.

Based on the results shown in Table 2, we used primers P2

(forward), P34 (reverse), and probe on the examination of 16S

rRNA M. leprae using real time PCR.

Sampling was done in the dressing room of the Derma-

tovenereology Laboratory, Dr. Soetomo Hospital. RT-PCR tests

were performed at the Leprosy Study Group, Institute of

Tropical Disease, Universitas Airlangga, Surabaya, Indonesia

and immunohistochemical examination was performed at

Gramik Laboratory, Faculty of Medicine, Universitas

Airlangga. The study was conducted from September 2006

to December 2006. Research proposal had been approved by

the Ethics Commission of the Dr. Soetomo Hospital (ethical

clearance number 59/Panke.KKE/2005).

Assessment of the expression of Rab5, Rab7, TACO,

Lep-LAM, and PGL-1 was done by a combination of analytical

calculation of the number of cells counted manually in 20

visual fields and image analysis method to calculate the

intensity of immunohistochemical staining using Nikon

Capture NX (Nikon Corporation, Minato, Tokyo, Japan) [18].

Data processing and analysis

A reliability test was done between two observers in counting

the number of macrophage cells expressing Rab5, Rab7,

TACO, Lep-LAM, and PGL-1. Score division to count the num-

ber of cells was based on quartiles and median. Score division

for staining intensity was based on the percentiles of 33% and

66%. The multiplication result was obtained from the score of

cells counted with the score for counting staining intensity in

each sample. Nonparametric two-sample comparison test on

the variables Rab5, Rab7, TACO, and PGL Lep-LAM-1 expres-

sion was based on the presence or absence of 16S rRNA of

M. leprae. Score division for the calculation of the quantity

of 16S rRNA of M. leprae was based on quartiles and median.

Nonparametric correlation test was done between the score

of 16S rRNA of M. leprae with a score of Rab5, Rab7, TACO,

Lep-LAM, and PGL-1.

Fig. 1 – Forward and reverse pr

Results

There were 47 patients who met the criteria, consisting of 30

men (63.83%) and 17 women (36.17%). To determine the viabil-

ity ofM. leprae, this study examined 16S rRNA ofM. lepraewith

real time PCR using the primers P2 (forward), P34 (reverse),

and probe Taq man, with the results as shown in Fig. 2.

The amplification plot in Fig. 2 shows the number of cycles

when fluorescence began to be detected in each sample. Vary-

ing results were seen. Of the 47 samples, we obtained positive

results in 35 samples (74.47%) and negative results in 12 sam-

ples (25.53%).

Figs. 3–7 show the representative results of immunohisto-

chemical examination of skin biopsies to detect Rab5, TACO,

Lep-LAM, PGL-1, and Rab7 by monoclonal antibodies against

each protein.

Reliability test results demonstrated the suitability of the

macrophage cell counts expressing Rab5, Rab7, TACO, Lep-

LAM, and PGL-1 in both observers (p > .05). Combined score

was obtained by multiplying the score of the number of cells

expressing Rab5, Rab7, TACO, Lep-LAM, and PGL-1 with a

score of staining intensity of cells expressing Rab5, Rab7,

TACO, Lep-LAM, and PGL-1. Mann–Whitney U test was per-

formed to determine differences in the expression of Rab5,

Rab7, TACO, Lep-LAM, and PGL-1 based on the viability of

the positive and negative (Table 3).

In Table 3 it can be seen that the expressions of Rab5, Rab7,

LEP LAM, and PGL-1 in macrophages with positive viability of

macrophages were significantly different compared with

those with negative viability (p < .05), whereas TACO expres-

sion in macrophages with positive viability was not signifi-

cantly different compared with that in macrophages with

negative viability (p > .05).

Distribution of scores based on the quartiles was per-

formed from the results of calculations of 16S rRNA M. leprae

quantity by real time PCR. Furthermore, the correlation test

between the combined scores of cell numbers and staining

intensity with a score of quantity of 16S rRNA showed results

as in Table 4.

In Table 4 it can be seen that the expression of Rab5, Rab7,

Lep-LAM, and PGL-1 in macrophages has a correlation with

the quantity of 16S rRNA M. leprae as a marker of viability

(p < .05), whereas expression of TACO lacks correlation with

the quantity of 16S rRNA of M. leprae (p > .05).

Discussion

The phagolysosome process in macrophages was studied by

immunohistochemical assay to examine the expression of

imers and probes at P2–P3.

Table 1 – Design of primers and probes using primer express software program.

Name Position Location (bp) Sequence (50–30)

P2 Forward 69–91 CGG AAA GGT CTC TAA AAA ATC TTF Forward 105–123 GAG TGG CGA ACG GGT GAG TP24 Forward 142–161 CCT GCA CTT CAG GGA TAA GCP25 Forward 150–169 TCA GGG ATA AGC TTG GGA AA

Probe 128–142 CGT GGG TAA TCT GCP3 Reverse 218–239 CAT CCT GCA CCG CAA AAA GCT TR Reverse 146–167 CAC TTC AGG GAT AAG CTT GGG AP34 Reverse 185–204 ATG CGC CTT GAA GTC CTA TCP35 Reverse 193–212 ACA AGA CAT GCG CCT TGA A

158 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 5 ( 2 0 1 6 ) 1 5 5 –1 6 3

Rab5, Rab7, TACO, Lep-LAM, and PGL-1 in the macrophages.

Theoretically, the host’s proteins, Rab5, Rab7, and TACO, as

well as M. leprae’s proteins, Lep-LAM and PGL-1, play a role

in the phagolysosome process through membrane trafficking,

which is a material transfer event from one organelle to

another organelle during a series of phagolysosome processes

[19].

Our study revealed the significant differences in Rab5

expression between thepositive viability andnegative viability

groups (p = .002; Table 3), whereas Rab5 expression profile in

Table 2 – The results of sensitivity tests of primer pairs P2–P3,

Primer pairs Basal pairs T

P2–P34 140 3P2–P35 148 3P2–P3 171 3

Fig. 2 – Reverse transcription-polymerase chain reaction amplifi

leprosy patients.

the viable group was higher than that in the nonviable group.

Rab5 accumulation in the viable group indicates the occur-

rence of membrane trafficking inhibition that causes the fail-

ure of M. leprae ingestion by phagolysosome, in accordance

with phagolysosome studies on M. tuberculosis and M. bovis

BCG, inwhich Rab5 expression inmacrophages reveals phago-

some maturation failure [20]. Extrapolation was done by the

authors on the base of character similarities of bothmycobac-

teria. Spearman’s correlation test between the scores of 16S

rRNA ofM. leprae and Rab5 (p = .001; Table 4), further strength-

P2–P34, and P2–P35.

hreshold cycle (CT) Minimal quantity (pg)

6.9 0.0019.44 0.14.9 1

cation plot of 16 subunit ribosomal RNA in skin biopsies of

Fig. 3 – Expression of Rab5 in the skin biopsy section of sample number 14. (A) With the enlargement of 100�; and (B) with the

enlargement of 450�. Note. N = negative reaction (any colors other than brown); P = positive reaction (brown).

Fig. 4 – Expression of tryptophan aspartate-containing coat protein in the skin biopsy section of sample number 3. (A) With

enlargement of 100�; and (B) with enlargement of 450�. Note. N = negative reaction (any colors other than brown); P = positive

reaction (brown).

Fig. 5 – Expression of leprae lipoarabinomannan in the skin biopsy section of sample number 8. (A) With a magnification of

100�; and (B) with a magnification of 450�. Note. N = negative reaction (any colors other than brown); P = positive reaction

(brown).

I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 5 ( 2 0 1 6 ) 1 5 5 –1 6 3 159

ens the role of Rab5 through membrane trafficking that is

responsible for phagolysosome failure due to a significant cor-

relation between the expression of Rab5with the expression of

16S rRNA of M. leprae as a marker of M. leprae viability.

In this study, using the Mann–Whitney U test, we also

found significant differences in Rab7 expression between

the viable and nonviable group (p < .001; Table 3), where the

profile of Rab7 expression in the viable group was higher than

that in the nonviable group. The presence of Rab7 expression

in macrophages containing living bacteria where there was

phagolysosome failure denied the theory that the failure of

phagosome maturation continuing in the phagolysosome

Fig. 7 – Expression of Rab7 in the skin biopsy section of sample number 6. (A) With enlargement of 100�; and (B) with

enlargement of 450�. Note. N = negative reaction (any colors other than brown); P = positive reaction (brown).

Fig. 6 – Expression of phenolic glycolipid-1 in the skin biopsy section of sample number 16. (A) With the enlargement of 100�;

and (B) with the enlargement of 450�. Note. N = negative reaction (any colors other than brown); P = positive reaction (brown).

160 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 5 ( 2 0 1 6 ) 1 5 5 –1 6 3

occurs due to failure of Rab7 recruitment. It can also be

assumed that there are Rab7 receptors in the phagosome,

not only in the late endosome. Phagolysosome studies in

other intracellular bacteria, Legionella pneumophila and M.

tuberculosis, showed confirming results in which there was

Rab7 expression on macrophages that contained viable bacte-

ria and no Rab7 expression in macrophages containing dead

bacteria. Similar properties of M. leprae with M. tuberculosis

were also shown in studies on the phagolysosome in M. tuber-

culosis where the macrophages with viable bacteria exhibited

Rab5 and Rab7 expression [21–23]. In addition, in studies

using latex bead, with the provision of phosphoinositide

Table 3 – Mann–Whitney U test results of the differences inthe expression of Rab5, Rab7, tryptophan aspartate-con-taining coat protein (TACO), leprae lipoarabinomannan(Lep-LAM), and phenolic glycolipid-1 (PGL-1) based onpositive and negative viability with p = .05.

Variables p

Rab5 .002Rab7 <.001TACO .584Lep-LAM <.001PGL-1 <.001

3-kinase (PI3K) inhibitor wortmannin which inhibits the

phagolysosome process, the accumulation of Rab7 was found

in macrophages, while the successful phagolysosome did not

show the expression of Rab7 [11]. This suggests the possibility

of an independent PI3K pathway to the success of the

phagolysosome process. Spearman’s correlation test between

the scores of 16S rRNA of M. leprae and Rab7 (p < .001; Table 4)

supports the fact that Rab7 expression correlated significantly

with the expression of 16S rRNA of M. leprae as a marker of M.

leprae viability. This study suggests that in M. leprae infection

where phagolysosome failure is characterized by high Rab5

Table 4 – Spearman correlation test between combinedscores between cell number and staining intensity of theexpression of Rab5, Rab7, tryptophan aspartate-containingcoat protein (TACO), leprae lipoarabinomannan (Lep-LAM),and phenolic glycolipid-1 (PGL-1) with the score of 16subunit ribosomal RNA M. leprae quantity (p = .05).

Variables Correlation coefficient p

Rab5 0.483 .001Rab7 0.682 <.001TACO 0.065 .662Lep-LAM 0.608 <.001PGL-1 0.491 <.001

I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 5 ( 2 0 1 6 ) 1 5 5 –1 6 3 161

and Rab7 expressions in macrophages, Rab5 and Rab7 can be

used as markers of M. leprae viability.

LAM in mycobacteria has the ability to bind phosphatidyli-

nositol to the cellwall and is located at the surface ofmycobac-

teria. LAM also has an ideal position for initial interaction

between mycobacteria and macrophages [13,24]. Lep-LAM,

derived from M. leprae, has a structure similar to that in

mannosylated-LAM (Man-LAM), so it is thought to have the

ability to inhibit the bactericidal process by macrophages and

allows extrapolation of the Man-LAM role in phagolysosome

fusion failure. Although Lep-LAM contains fewer mannoses

than Man-LAM, Lep-LAM is found in greater numbers in M.

leprae compared with the number of Man-LAM in M. tuberculo-

sis. This causes Lep-LAM to remain virulent particularly in the

inhibition of phagolysosome fusion, resulting in M. leprae per-

sistence inmacrophages [14]. In this study, theMann–Whitney

U test found significant differences in Lep-LAM expression

between the viable and nonviable groups (p < .001), where the

Lep-LAM expression on macrophages in the viable group was

higher than that in nonviable group (Table 3). Lep-LAM accu-

mulation inmacrophageswith positive viability demonstrates

the role of Lep-LAM through the membrane trafficking barrier

that occurs due to the inhibition of Ca2+ increase. Additionally,

Lep-LAM is thought to have properties similar to Man-LAM

that can inhibit the Ca2+ channel in the endoplasmic reticulum

that would cause phagolysosome failure. Spearman’s correla-

tion test between 16S rRNA M. leprae scores and Lep-LAM

(p < .001; Table 4), reinforces the fact that the expression of

Lep-LAM has a significant correlation with the expression of

16S rRNA of M. leprae as a marker of M. leprae viability. Thus,

the expression profile of Lep-LAM in M. leprae infection can

be used as a viability marker, based on the role of membrane

trafficking where there has been an inhibition of increased

Ca2+ required to destroy germs.

PGL-1, a cell wall component of M. leprae, is associated

with the ability of this bacterium to survive. In the M. tubercu-

losis phagolysosome process, PGL is hypervirulent [25]. Inhibi-

tion of the phagolysosome process by PGL-1 in M. leprae is

suspected to occur through the inhibition of the lysosomal

activation process pathway [26]. In this study, we revealed sig-

nificant differences in PGL-1 expression between the viable

and nonviable group (p < .001), whereas PGL-1 expression in

the viable group is higher than that in the nonviable group

(Table 3). This proves that the lysosomal activation pathway

barriers play a role in the failure of the phagolysosome infect-

ing M. leprae. Spearman’s correlation test between 16S rRNA

scores of M. leprae and PGL-1 (p < .001; Table 4) further

strengthens the role of PGL-1 through a pathway that is

responsible for phagolysosome failure because there is a cor-

relation between the mean PGL-1 expression with the expres-

sion of 16S rRNA of M. leprae as a marker of viability. The

research was supported by a study conducted by Cho et al.

[27], who carried out an evaluation after treatment was given.

They found a significant reduction of PGL-1 antigen in blood.

In addition, research by Ramasesh et al. [28] also showed that

only viable M. leprae could produce PGL-1 where there was no

PGL-1 biosynthesis in uninfected macrophages. PGL-1 was

thought to directly inhibit proteolytic enzymes in the lyso-

somes, causing M. leprae to survive. However, another study

conducted by Hashimoto et al. [12] led to the conclusion that

PGL-1 did not show any correlation with the viability of M.

leprae as PGL-1 could still be found in the patient’s body,

although many had been damaged [12]. The remaining pres-

ence of PGL-1 in the skin tissue of leprosy patients who had

received treatment was because of the possibility that PGL-1

molecules could not be immediately degraded, although the

bacteria were dead. The results of this study showed that

the profile of PGL-1 expression in M. leprae infection could

be used as a marker of viability.

TACO is a protein that is bound by actin forming microfil-

ament structures that function for the movement and distri-

bution of organelles, including phagosome maturation in the

phagolysosome process of shrinkage (ruffling), chemotaxis,

and phagocytosis. TACO facilitates intracellular infection by

inhibiting the formation of actin so that the movement of

bacteria-containing phagosomes and the occurrence of

fusion with other intracellular vesicles becomes disrupted

[20,29]. In this study, a Mann–Whitney U test found no signif-

icant difference in TACO expression between the positive via-

bility group and the negative viability group (p = .584; Table 3).

Spearman’s correlation test between 16S rRNA of M. leprae

scoreswith TACO (p = .662) also showed no significant correla-

tion (Table 4). Thus, TACO, through the inhibition of

phagolysosome fusion pathway (ruffling), does not play a role

in the failure of M. leprae phagolysosome. It is not in accor-

dance with a phagolysosome study conducted in M. bovis

BCG where there is TACO accumulation in the phagosome

membrane that acts as a barrier to phagolysosome fusion,

thereby increasing the survival of germs. In the study with

other intracellular bacteria, namely Helicobacter pylori, there

is indication of phagolysosome fusion interruptions caused

by the retention of TACO [11]. In this study, TACO retention

of successful phagolysosomes can be caused by the presence

of a compound that accumulates in macrophages and influ-

ences TACO retention. A study in Japan also showed that

the accumulation of TACO in phagosome membranes was

not associatedwith the presence or absence of viableM. leprae

in macrophages [30]. In an in vitro study conducted by Suzuki

et al. [30], macrophages found with a negative smear exami-

nation showed TACO expression with moderate to vigorous

quality. The fact from Suzuki et al.’s study [30] can lead to

an assumption that TACO is not only retained by viable M.

leprae, but also by deadM. leprae. In addition, macrophage cul-

tures introduced with M. leprae showed no TACO expression,

so it can also be interpreted that TACO is not likely to be

involved in the mechanism of bacterial killing. Similarly, the

amount of lipid that fills the phagosome causes M. leprae to

float, so contact between TACO with M. leprae is reduced.

The unlikely role of TACO in phagolysosome process failure

was also indicated in in vivo studies by Suzuki et al. [30] in

which the macrophages derived from skin biopsies of

patients with lepromatous leprosy and tuberculoid type

showed that TACO expressions were not significantly differ-

ent. The above explanation indicates TACO has no role in

phagolysosome failure of M. leprae infection. In other words,

other phagocytic pathways that play a role in phagocytosis

process of M. leprae and TACO expression profiles cannot be

used as a marker of M. leprae viability.

In this study it can be assumed that the phagolysosome

process of M. leprae infection can also be carried out through

162 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 5 ( 2 0 1 6 ) 1 5 5 –1 6 3

an independent PI3K pathway, andwhen the presence of Rab7

expression in phagolysosome fails, there is a possible pres-

ence of a Rab7 receptor in the phagosome. The presence of

Lep-LAM can inhibit these pathways, thereby causing Rab7

to accumulate permanently in the macrophages. This sug-

gests that the PI3K-dependent pathway is not the only alter-

native for the M. leprae phagolysosome process. The

possibility that the PI3K pathway is independent in this

phagolysosome has been proven by studies conducted by

Vieire et al. [11], while the presence of Rab7 receptor in phago-

some has been demonstrated by Clemens et al. [23]. The inhi-

bition of the phagolysosome process by PGL-1 in M. leprae is

suspected to run through the inhibition of lysosomal activa-

tion pathway. The process is in line with some research that

has been conducted on M. tuberculosis. TACO does not play a

role in the failure of the phagolysosome in M. leprae infection.

In other words, other phagocytic pathways that play a role in

the process of phagocytosis of M. leprae and TACO expression

profiles cannot be used as markers of M. leprae viability.

An important finding as the result of this study is the role

of membrane trafficking in phagolysosome failure, which is

represented by two compounds derived from the host (Rab5

and Rab7) and from the agent (Lep-LAM). In addition, we

found the role of PGL-1 in the inhibition of lysosomes activa-

tion pathway in phagolysosome failure. Therefore, the

expression profiles of Rab5, Rab7, Lep-LAM, and PGL-1 can

be used as markers of M. leprae viability. From these findings,

an early diagnostic tool for leprosy based on expression pro-

files of Rab5, Rab7, Lep-LAM, and PGL-1 can be developed.

Early diagnosis of leprosy is a strong rationale for early ther-

apeutic intervention, so as to prevent the occurrence of dis-

ability or transmission. In addition to the above findings, it

is important to study the expression profiles of Rab5, Rab7,

Lep-LAM, and PGL-1 in peripheral blood mononuclear cells

(PBMCs). Based on the results of research conducted by the

Leprosy Study Group, Institute of Tropical Disease, Universi-

tas Airlangga [31], the results obtained no significant differ-

ences between expression profiles of 16S rRNA of M. leprae

in skin biopsy tissue and PBMCs using real time PCR. There-

fore, this study can be used as a base to explore the expres-

sion profiles of Rab5, Rab7, Lep-LAM, and PGL-1 in PBMCs,

which means that blood tests without skin biopsy are suffi-

cient to create a simplified and noninvasive early diagnostic

marker device for leprosy viability.

Conflicts of interest

The authors have nothing to disclose.

Acknowledgment

This study was presented at the International Congress of

Leprosy in Hyderabad, India in January 2008.

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