TITLE
ACID TOLERANCE AND INTRACELLULAR SURVIVAL OF BRUCELLA
ACID TOLERANCE AND INTRACELLULAR SURVIVAL OF BRUCELLA
Thomas A. Ficht
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University,
College Station, TX 77843-4467, USA
CURRENT STATUS OF BRUCELLA ERADICATION
Brucellosis in humans is increasing in developing areas of the Mediterranean region, Middle East,
western Asia and parts of Africa and Latin America. B. melitensis, the most pathogenic species in
humans, constitutes a public health priority. Although a notifiable disease, official figures do not reflect
the number of human infections that occur each year which may be as high as 10 and 25 times the
reported figures. The main cause for this underestimation is unrecognized and inaccurate diagnosis, i.e.,
"fever of unknown origin". In Mediterranean and Middle Eastern countries the annual incidence of
brucellosis in people varies from 1 to 78 cases per 100,000 and in some confined endemic areas lacking
animal control measures greater numbers of cases have been reported (1). For example, a recent survey
in a randomly selected human population in a country of the Arabic Peninsula revealed serological
evidence of exposure to Brucella close to 20%, with more than 2% of these having active disease.
Similar figures may be expected from most countries in which the disease is endemic in the animal
population. Historically, a higher seroprevalence of brucellosis may also be expected in occupationally
exposed groups.
Animal brucellosis poses a barrier to trade of animals and animal products and could seriously impair
socio-economic development, especially for livestock owners, which represent a vulnerable sector in
many rural populations. As an indication of the importance of this disease, plans to eliminate ovine,
caprine and bovine brucellosis from the European Union were expected to receive over half of the total
European Commission funding for animal disease control measures in 1997. In the U.S. the primary
brucellosis concern is the transmission of B. abortus from infected bison to cattle, and transmission
among wild-life (bison and elk) resulting from man-made feed grounds in the Western U.S designed to
attract elk and to enhance tourism. Continued investigation of Brucella pathogenesis is necessary
because i) brucellosis remains a major zoonosis worldwide and causes economic hardship as a result of
the loss of livestock (sheep, goats and cattle); ii) B. melitensis and B. suis represent emerging pathogens
in cattle, thus extending their opportunities to infect humans; iii) recent isolations of distinctive strains
from marine mammals is consistent with an extended ecological range (2,3), iv) molecular genetic
analysis has demonstrated a phylogenetic relationship to Agrobacterium, Phyllobacterium,
Ochrobactrum, and Rhizobium indicating that this information will have more general applicability, and
v) Brucella represents a continued threat as a weapon in biological warfare or bioterrorism until better
treatment regimens and preventative measures are developed (4).
SURVIVAL MECHANISMS
Brucella sp. are facultative intracellular pathogens and representatives of the 2-subdivision of the
proteobactereaceae, a group of organisms of agricultural and medical importance which includes
Agrobacterium, Phyllobacterium, Ochrobactrum, and Rhizobium. Biological differences with the
enterobactereaceae suggests the potential for adaptive differences and perhaps the evolution of unique
systems to deal with environmental stresses. The first and most obvious difference is the route of
infection. Brucella are thought to rapidly penetrate the oral mucosa and enter the lymphatic system and
blood stream (5). This is consistent with the observed acid sensitivity and aerobic metabolism of this
group of organisms. The energy yielding metabolic processes in Brucella are essentially oxidative.
Brucella cannot grow fermentatively and is not equiped to survive anaerobic conditions found in the
lower intestines. Survival in the bovine rumen, containing elevated amounts of organic acids, has never
been documented. Fermentative production of organic acids does not occur nor do they acidify the
growth medium. The absence of an environmental reservoir suggests specific adaptation to life within
the tissues of the host. As a result, the primary and perhaps only encounter of Brucella with acidic
environments may be within the phagocytic cell vacuole.
Initial survival and dissemination of Brucella is thought to be based on the inhibition of degranulation
and oxidative burst in neutrophils (PMNs) (6-8), and in macrophages (M?s) by inhibiting phagolysosomal
fusion (9,10). The first confirmed virulence factor expressed by Brucella is the lipopoysaccharide
(LPS) which protects the organism from complement-mediated lysis, as well as enhancing intracellular
survival (11). The organisms persist in the host for extended periods safely harbored within the
intracellular milieu. Intracellular survival has also been attributed to the inhibition of primary
degranulation in bovine PMNs by 5’GMP and adenine, and fusion in murine macrophages by undefined
components in water-soluble extracts from whole Brucella (10,12,13). The inability of purE mutants to
survive intracellularly was attributed to limited purine levels present in the phagosomal compartment
(14). Unfortunately, no attempt was made to examine P-L fusion in infected M?s using these mutants.
The killing activity of oxidative compounds may be ameliorated by the production of superoxide
dismutase and/or catalase, although mutants in these genes have little demonstrable affect on
intracellular survival (15). Recent evidence from two laboratories has suggested that Brucella
containing phagosomes acidify to pHsbetween 4.0 and 4.5 (16,17). These data would appear to indicate
that acidification of the phagosomal compartment proceeds despite the inhibition of P-L fusion, and that
the two are separate, but overlapping mechanisms (18,19).
The basic premise of current work in our laboratories is that resistance to acid conditions in either the
presence or absence of P-L fusion represents a primary determinant for intracellular survival of Brucella.
Experiments are in progress to characterize acid tolerance mechanisms required for intracellular survival
and to characterize the genetic components involved in order to develop vaccine strains which are unable
to transfer between hosts. Genetic characterization of the mechanisms required for long-term
intracellular survival should provide insight into mechanisms active in genetically intractable obligate
intracellular pathogens such as the chlamydia and erlichia.
ACID TOLERANCE IN BRUCELLA
Early reports of acid tolerance in Brucella indicated a level of sensitivity exceeding that observed for
E. coli or S. flexneri (20), and medical opinion based on anecdotal evidence at the turn of the century
suggested the discontinued use of antacids when traveling in Brucella endemic regions (21). The
relationship between acid environment and intracellular survival of Brucella was only recently
investigated by Detellieaux, et al who revealed that inhibition of endosome acidification blocked
B. abortus replication following uptake into Vero cells (18). A result reminiscent of reports indicating
the importance of compartmental acidification in the survival of S. typhimurium (19). More recent
results have indicated the persistence of Brucella within acidic macrophage compartments with
pHsbetween4.0 and 4.5 (16,17). These results would suggest that the Brucella does not require the
systems present in the enterobactereacea which protect those organisms from the extreme pHs found in
the digestive system. Brucella may represent a simplified paradigm expressing only those mechanisms
necessary for intracellular survival.
VETERINARY PATHOBIOLOGY
5
THOMAS A. FICHT
Table 1
Comparison of acid tolerance mechanisms in Brucella and Salmonella
Acid Shock
Stationary
Brucella
Salmonella
Fig.1 Acid tolerance responses
in B. abortus plotted
as a functionBrucella
of growth stage inSalmonella
complex
medium (trypticase
soy broth).pH5.8
The solid line represents
the growthPre-acid shock
(homeostasis)
curve
(turbidity
or
colony
forming
units)
for
B.
abortus.
The
shaded
areas
revealAcid shock
pH4.4
pH4.5
the relative protection?68g
from acid shock 50
(2 hours @ 2-10 x +i
109 cfu/ml in TSB
ASPs
+
adjusted to pH 3.4 with hydrochloric acid). Although it is possible that the
Complex medium
+
+
+
+
stationary phase acid adaptation persists following transfer to freah medium,
MinimalCBP
medium
- response, albeitND
24 mutants expressed -the stationary phase
reduced, while the+
RpoS dependenta
NDh
+ early-log repsonseND
was undetectable.+/Cross protectionb
ND
ND
+
furc
+
ND
organic acidsd
ND
+
ND
+
PhoPe
+
ND
RecAf
ND
+
ND
a. rpoS is defective in a number of fortuitously isolated attenuated strains of S. typhimurium,
including LT2, aa transient ATR has been characterized in these rpoS mutants (22,23)
b. functions essential to pH homeostasis (cytoplasmic buffering) are activated along with functions
altering membrane hydrophobicity, tolerance to polymixin B, heat and salt stresses (24)
c. Fur binding was shown to regulate the expression of 14 ASPs in an iron-independent fashion
d. RpoS is essential for protection against weak (organic acids)
e. PhoP has been reported to be an ASP used for protections against low pH (25)
f. DNA repair systems are also required to resist acid stress (26)
g. Rafie-Kolpin, et al (27).
h. Not determined
I. CBP24 is required.
Recent results from our laboratory suggest that at least three mechanisms contribute to acid tolerance in
B. abortus. The first mechanism is referred to as log-phase, acid shock adaptation and is induced by
exposure to reduced pH=4.4 during early-log phase growth (2 x 109 CFU/ml). Acid shock adaptation
was not observed in cells grown in minimal medium and was no longer inducible in cells in rich media
at mid-log growth phase presumably as a result of the induction of the stationary phase response (next
paragraph) (28). Although similar to Salmonella in expression during early-log growth phase, the lack
of expression in minimal media is reminiscent of the AR system of E. coli which involves amino acid
decarboxylation (29). Inhibition of protein synthesis just prior to acid shock conditions prevents the
development of acid tolerance. A second acid inducible system was revealed during the stationary
growth phase by a mutation in CBP24 a calcium binding protein which is an acid shock protein (ASP).
Acid shock adaptation observed in Brucella during early log growth phase is replaced during mid-log
growth phase (5 x 109 CFU/ml) probably as a result of stationary phase induction. Stationary phase is
generally associated with increased resistance to a number of environmental stresses and it is commonly
believed that increased resistance to stress associated with stationary phase enhances survival and/or
virulence (30). A stationary phase sigma factor RpoS which is induced in the enterobactereaceae by
many of the growth conditions common during stationary phase (i.e., limited nutrients, limited oxygen,
reduced pH and toxic by-products including weak acids) has not been identified in Brucella . Stationary
phase ATR in S. typhimurium is superior to log phase sustained ATR, protecting cells for longer periods
of time at pH3 (31). An RpoS independent ATR in stationary phase has also been described in
S. typhimurium (32).
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THOMAS A. FICHT
Several Brucella acid shock proteins have been documented (27), but only a handful have been
characterized (33). The first, GroEL is not restricted to acid shock alone and is a major heat shock
protein Hsp65. Variation in the electrophoretic mobility of this protein suggests covalent modification
may accompany expression under acid shock conditions. The second, CBP24 is a calcium binding protein
which appears to have a specific role in acid shock adaptation. Mutants defective in CBP24 lack acid
shock adaptation and are defective in the stationary phase acid tolerance response. These mutants did
reveal and acid-inducible acid tolerance system was functional during stationary phase, but is
presumably masked by the normal stationary phase response. The second gene product, CYD oxidase,
catalyzes the terminal step of the alternative electron transport chain. CYD deficient mutants are
extremely sensitive to reduced pH (28). The acid sensitivity observed in CYD oxidase mutants may be
attributable to a lack of overall pH homeostasis, similar to that observed in H+-ATPase in E. coli (next
section). As a result it is not possible to characterize acid shock adaptation in this strain. The role of an
RpoS like gene product can only be inferred from the stationary phase response described above.
Mutants defective in a phoP-homolog and fur had no apparent effect on acid shock adaptation and the
effect on the other mechanisms remains undetermined(28). Protection against organic acids remains to
be elucidated. Although there are high concentrations of these substances in the bovine rumen, Brucella
infection in ruminants does not appear to involve rumen transit.
ROLE OF ACID TOLERANCE IN VIRULENCE
The contribution of acid tolerance to virulence has been extensively studied in the enterobactereaceae
(34). Although a strong correlation exists it has been difficult to establish a direct connection.
Salmonella mutants defective in the major proton translocating ATPase (atp), are unable to express the
pH 5.8 homeostasis system. In its absence, inducible homeostasis systems are unable to keep up with
the intracellular proton burden and cell viability is reduced as a result of sensitivity of several systems to
reduced pHi (30). Mutations in other ATR loci have revealed little dramatic effect on virulence, and in
fact, only modest effects on ATR. For example, mutants in the ferric uptake regulator (fur) exhibited
attenuated phenotypes (1-2 logs), but attenuation varied depending on the genetic background of the
S. typhimurium, perhaps as a result of redundancies inherent in the system(30,34). Consistent with this
possibility, introduction of multiple mutations into a single strain has been shown to drastically reduce
ATR and virulence (34).
Mutants defective in the expression of CBP24 exhibit reduced survival in the mouse model
(approximately 2 logs lower than wild-type). It is presumed, although not proven, that this protein is
expressed in order to sequester calcium under these conditions. In support of this hypothesis, B.abortus
has been shown to excrete calcium under low pH conditions (28). Survival of mutants defective in CYD
oxidase was greatly attenuated in the mouse model (no organisms were recovered 2-3 weeks postinoculation).
Preliminary interpretation of these results would suggest that the acid-inducible, stationary phase system
expressed in CBP24 mutants, but not in CYD mutants is most important to intracellular survival.
However, mutation in CYD oxidase like the H+-ATPase in E. coli may affect overall proton homeostasis
and the sensitivity to other activities which affect proton balance. For these reasons we have undertaken
an effort to more fully characterize the contribution of acid tolerance to intracellular survival along with
the identification of additional acid tolerance gene products.
INTRACELLULAR VS. EXTRACELLULAR ACID TOLERANCE
In addition to the ATR response, additional mechanisms have been characterized in other members of
the enterobactereaceae. There is little likelihood of identifying such systems in Brucella, since they
appear to function at pH extremes not experienced by Brucella or in conjunction with anaerobic
VETERINARY PATHOBIOLOGY
5
THOMAS A. FICHT
environments in which Brucella cannot survive . Acid resistance (AR) in E. coli and Shigella provide
protection down to pH2.5 (31,35,36), which may be more important for survival during transit of the
stomach and duodenum. Two of the three AR systems identified are active during fermentative
metabolism (31). The method by which these systems protect the cell is similar to pH homeostasis
described for Salmonella using lysine decarboxylase. Decarboxylation of the amino acids consumes a
proton and subsequent excretion of the product via a membrane antiporter is coupled with the
development of an electrogenic potential (proton motive force) used to take up the amino acids (29,37).
VETERINARY PATHOBIOLOGY
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THOMAS A. FICHT
FUTURE EXPERIMENTATION
Further experimentation is required in order to clarify the conof acid tolerance to survival and virulence.
The work being performed in my laboratory is designed to achieve the following specific aims,
1. Characterize acid shock adaptation and stationary phase acid tolerance responses in B. abortus.
2. Characterize the role of individual genes in survival and virulence
3. Characterize the contribution to acid shock adaptation and stationary phase acid tolerance to
intracellular survival and virulence.
The goal of the experiments proposed is to mechanistically, biochemically and genetically characterize
the acid tolerance mechanisms of B. abortus and their contribution to survival and pathogenesis. Based
on our preliminary observations demonstrating the complete absence of acid tolerance in CYD oxidase
mutants and its lack of intracellular survival we hypothesize that acid tolerance mechanisms are
contributing factors to intracellular survival and virulence in Brucella. Characterization of the acid
tolerance mechanisms and the timing of their expression will provide valuable clues to their role during
infection and will help in the construction of mutant strains of varying attenuation to be used as vaccine
strains in several different host species.
BIBLIOGRAPHY
1. Corbel, M. J. 1997. Brucellosis: An Overview. Emerg.Inf.Dis. 3:213-221.
2. Ewalt, D. R., J. B. Payeur, B. M. Martin, D. R. Cummins, and W. G. Miller. 1994.
Characteristics of a Brucella species from a bottlenose dolphin (Tursiops truncatus).
J.Vet.Diagn.Invest. 6:448-452.
3. Miller, W. G., L. G. Adams, T. A. Ficht, N. Cheville, J. Payeur, D. Harley, and S. Ridgeway.
1998. First case report of Brucella abortion in a bottlenose dolphin Tursiops truncatus. J.Zoo
Wildl.Med. in press
4. Kaufmann, A. F., M. I. Meltzer, and G. P. Schmid. 1997. The economic impact of a bioterrorist
attack: Are prevention and postattack intervention programs justifiable? Emerg.Inf.Dis. 3:83-94.
5. Epstein, L. D., A. Munoz, and D. He. 1996. Bayesian Imputation of Predictive Values When
Covariate Information is Available and Gold Standard Diagnosis is Unavailable. Stat.in Med.
15:463-476.
6. Riley, L. K. and D. C. Robertson. 1984. Ingestion and intracellular survival of Brucella abortus in
human and bovine polymorphonuclear leukocytes. Infect.Immun. 46:224-230.
7. Bertram, T. A., P. C. Canning, and J. A. Roth. 1986. Preferential inhibition of primary granule
release from bovine neutrophils by a Brucella abortus extract. Infect.Immun. 52:285-292.
8. Kreutzer, D. L., L. A. Dreyfus, and D. C. Robertson. 1979. Interaction of polymorphonuclear
leukocytes with smooth and rough strains of Brucella abortus. Infect.Immun. 23:737-742.
9. Harmon, B. G., L. G. Adams, and M. Frey. 1988. Survival of rough and smooth strains of Brucella
abortus in bovine mammary gland macrophages. Am.J.Vet.Res. 49:1092-1097.
10. Frenchick, P. J., R. J. F. Markham, and A. H. Cochrane. 1985. Inhibition of phagosomelysosome fusion in macrophages by soluble extracts of virulent Brucella abortus. Am.J.Vet.Res.
46:332-335.
VETERINARY PATHOBIOLOGY
5
THOMAS A. FICHT
11. Allen, C. A. and T. A. Ficht. 1997. Transposon Derived Brucella abortus Rough Mutants are
Attenuated and Exhibit Reduced Intracellular Survival. Infect.Immun. 65:1008-1016.in press
12. Canning, P. C., J. A. Roth, and B. L. Deyoe. 1986. Release of 5'-guanosine monophosphate and
adenine by Brucella abortus and their role in the intracellular survival of the bacteria. J.Infect.Dis.
154:464-470.
13. Bertram, T., P. Canning, and J. Roth. 1986. Preferential inhibition of primary granule release
from bovine neutrophils by a Brucella abortus extract. Infect.Immun. 52:285-292.
14. Drazek, E. S., H. H. Houng, R. M. Crawford, T. L. Hadfield, D. L. Hoover, and R. L. Warren.
1995. Deletion of purE attenuates Brucella melitensis 16M for growth in human monocyte-derived
macrophages. Infect.Immun. 63:3297-3301.
15. Latimer, E., J. Simmers, N. Sriranganathan, R. M. Roop,II, G. G. Schurig, and S. M. Boyle.
1992. Brucella abortus deficient in copper/zinc superoxide dismutase is virulent in BALB/c mice.
Microb.Pathog. 12:105-113.
16. Rouot, B. 1997. Acidification of phagosomes containing Brucella suis, a phenomenon independent
of phagosome-lysosome fusion is essential for intracellular multiplication. Brucellosis Res.Conf.
50:in press
17. Phillips, R. W., G. Buczynski, G. T. Robertson, J. Cardelli, and R. M. Roop,II. 1997.
Acidification of murine peritoneal macrophage phagosomes that contain Brucella abortus.
Brucellosis Res.Conf. 50:(Abstract)
18. Detilleux, P. G., S. L. Deyoe, and N. F. Cheville. 1991. Effect of endocytic and metabolic
inhibitors on the internalization and intracellular growth of Brucella abortus in Vero cells.
Am.J.Vet.Res. 52:1658-1664.
19. Alpuche-Aranda, C. M. A., J. A. Swanson, W. P. Loomis, and S. I. Miller. 1992. Salmonella
typhimurium activates virulence gene transcription within acidified macrophage phagosomes.
Proc.Natl.Acad.Sci.USA 89:10079-10083.
20. Garrod, L. P. 1937. The susceptibility of different bacteria to destruction in the stomach.
J.Pathol.and Bacteriol. 45:473-474.
21. Editorial, 1978. Antacids and brucellosis. Br.Med.J. 1:739-740.
22. Foster, J. W. 1993. The acid tolerance response of Salmonella typhimurium involves transient
synthesis of key acid shock proteins. Journal of Bacteriology 175:1981-1987.
23. Lee, I. S., J. Lin, H. K. Hall, B. Bearson, and J. W. Foster. 1995. The stationary-phase sigma
factor sigma S (RpoS) is required for a sustained acid tolerance response in virulent Salmonella
typhimurium. Molecular Microbiology 17:155-167.
24. Leyer, G. J. and E. A. Johnson. 1993. Acid adaptation induces cross protection against
environmental stresses in Salmonella typhimurium. Appl.Environ.Microbiol. 59:1842.
25. Bearso, S., B. Bearson, and J. W. Foster. 1997. Acid stress responses in enterobacteria. FEMS
Microbiol.Lett. 147:173-180.
26. Foster, J. W. 1995. Low pH adaptation and the acid tolerance response of Salmonella typhimurium.
Crit.Rev.Microbiol. 21:215-237.
27. Rafie-Kolpin, M., R. C. Essenberg, and J. H. Wyckoff,III. 1996. Identification and comparison of
macrophage-induced proteins and proteins induced under various stress conditions in Brucella
abortus. Infect.Immun. 64:5274-5283.
28. Ficht, T. 1998. Unpublished results. Infect.Immun.
VETERINARY PATHOBIOLOGY
5
THOMAS A. FICHT
29. Waterman, S. R. and P. L. Small. 1996. Identification of sigma S-dependent genes associated with
the stationary-phase acid-resistance phenotype of Shigella flexneri. Molecular Microbiology 21:925940.
30. Portillo, F. G. -d, J. W. Foster, and B. B. Finlay. 1993. The role of acid tolerance response genes
in Salmonella typhimurium virulence. Infect.Immun. 61:4489-4492.
31. Lin, J., J. Frey, J. L. Slonczewski, and J. W. Foster. 1995. Comparative analysis of extreme acid
survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. J.Bacteriol. 177:40974104.
32. Lee, I. S., J. L. Slonczewski, and J. W. Foster. 1994. A low-pH-inducible, stationary-phase acid
tolerance response in Salmonella typhimurium. J.Bacteriol. 176:1422-1426.
33. Lin, J. and T. A. Ficht. 1995. Protein synthesis in Brucella abortus induced during macrophage
infection. Infect.Immun. 63:1409-1414.
34. Wilmes-Riesenberg, M. R., B. Bearson, J. W. Foster, and R. Curtiss,III. 1996. Role of acid
tolerance response in virulence of Salmonella typhimurium. Infect.Immun. 64:1085-1092.
35. Gorden, J. and P. L. C. Small. 1993. Acid resistance in enteric bacteria. Infect.& Immun. 61:364367.
36. Small, P., D. Blankenhorn, D. Welty, E. Zinser, and J. L. Slonczewski. 1994. Acid and base
resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH. J.Bacteriol.
176:1729-1737.
37. Waterman, S. R. and P. L. Small. 1996. Characterization of the acid resistance phenotype and rpoS
alleles of shiga-like toxin-producing Escherichia coli. Infect.Immun. 64:2808-2811.
VETERINARY PATHOBIOLOGY
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THOMAS A. FICHT