Plasmid DNA from Yersinia pestis

Details Plasmid DNA from Yersinia pestis Plasmid DNA from Yersinia pestis

1999-09-30

2004-03-16

Fredman, Jeffrey (Department: 1634)

Chemistry: molecular biology and microbiology

Vector, per se

C435S252300, C536S023100

Reexamination Certificate

active

06706522

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
BACKGROUND OF THE INVENTION
Over the centuries, the bubonic plague (also known as the Black Death) has claimed the lives of millions of people. The disease is characterized by chills, fever, vomiting, diarrhea, painful swollen lymph nodes (buboes), blackening of the skin caused by ruptured blood vessels, and a very high mortality rate (up to 75% if left untreated). Treatment with antibiotics in the early stages of the infection is generally effective.
Bubonic plague is caused by the bacterium
Yersinia pestis
, which is transmitted to humans from rats or other rodents by fleas that feed on infected rodents and then bite humans. Reservoirs of the bacteria persist today, and attempts to eliminate wild rodent plague have proven ineffective. Occasional outbreaks of the deadly disease continue to occur, particularly in small towns, villages, and rural areas in developing countries.
While bacteria carry genetic material in their chromosomes, bacteria also often carry genetic material in loops of DNA called plasmids. Bacterial plasmids are nonessential, extrachromosomal genetic elements capable of autonomous replication. The genetic material in plasmids often encodes functions required for maintenance of the plasmid in its bacterial host and sometimes encodes optional functions that promote survival of the bacterial host under certain environmental conditions. Pathogenicity determinants are commonly plasmid-encoded, and fall within the category of optional plasmid-encoded functions.
Yersinia pestis
is a facultative intracellular parasite which harbors at least three different plasmids, designated pCD1, pPCP1, and pMT1, which are necessary for full virulence of the organism. One of the plasmids, designated pCD1, is also found in the enteropathogenic species
Yersinia pseudotuberculosis
and
Yersinia enterocolitica
(Ferber, et al.
Infect. Immun
. 31:839-841, 1981; Portnoy, et al.
Curr. Topics Microbiol. Immunol
. 118:29-51, 1985), whereas pMT1 and pPCP1 are unique to
Y. pestis
(Brubaker.
Clinical Microbiol Rev
. 4:309-324, 1991). Plasmids pMT1 and pPCP1 are thought to promote deep tissue penetration by
Y. pestis
and to contribute to the acute infection associated with this species. The
Y. pestis
genome shares much homology with that of
Y. pseudotuberculosis
, (Bercovier, et al.
Curr. Microbiol
. 4:225-229, 1980; Moore, et al.
Inter. J. Sys. Bacteriol
. 25:336-339, 1975), yet the infection caused by the latter organism is usually mild and self limiting (Butler, Plague and other yersinia infections, p. 111-159. In W. B. Greenbugh III and T. C. Merigan (eds.), Current topics in infectious disease, Plenum Press, New York, N.Y.,
1983).
An understanding of the differences in the pathogenesis of
Y. pestis
and
Y. pseudotuberculosis
may be afforded by comparing polynucleotide sequences or genes found on pMT1 or pPCP1 plasmids, and which are unique to
Y. pestis
. It has been found that
Y. pestis
strains lacking the pCD1 plasmid are completely avirulent. Therefore, determination of the complete pCD1 sequence may provide important information about the role of the plasmid in virulence in various pathogenic yersiniae.
The 9.5 kb plasmid pPCP1 encodes a bacteriocin termed pesticin, a pesticin immunity protein and a plasminogen activator activity. Loss of this plasmid increases the LD
50
of the organism by a factor of one hundred thousand, as measured by subcutaneous injection in the mouse model. (Sodeinde, et al.
Science
258:1004-1007, 1992).
The second plasmid unique to
Yersinia pestis
, designated pMT1, is a 100 kb plasmid that encodes the capsular protein Fraction 1 and the murine toxin (Protsenko, et al.
Genetika
19:1081-1090, 1983). The genes for the capsular proteins have been cloned and sequenced using
Y. pestis
strain EV76 (Galyov, et al.
FEBS Lett
. 277:230-232, 1990; Galyov, et al. 286:79-82, 199.1; Karlyshev et al.
FEBS Lett
. 305:37-40, 1992). The role of these proteins in plague pathogenesis has not been unequivocally determined, and the effect of mutational loss of these proteins on the LD
50
varies, depending on the animal model and route of infection (Brubaker
Curr. Top. Microbiol
. 57:111-118, 1972; Brubaker Rev. Infect. Diis. 5: S748-S758, 1983). However, pMT1 does appear to contribute to the acute phase of plague infection, as evidenced by a reduced morbidity associated with infection by strains lacking pMT1 (Drozdov, et al.
J. Med. Microbiol
. 42:264-268, 1995; Samoilova, et al.
J. Med. Microbiol
. 45:44.0-444, 1996;Welkos, et al.
Contrib. Microbiol. Immunol
. 13:229-305, 1995).
Information pertaining to the genetic characterization of the pMT1 molecule is limited. The size of the plasmid has been found to vary, either from variations in the versions of the plasmids or in technique to measure the plasmids, from 90 kb to 288 kb (Filippov, et al.
FEMS Microbiol. Lett
. 67:45-48, 1990). It is known that pMT1 is an integrative plasmid capable of integrating into
Y. pestis
chromosome with high frequency and at multiple sites, with integration likely resulting from IS100 homology between the plasmid and chromosome (Protsenko, et al.
Microbiol. Pathogen
11:123-128, 1991).
Previous characterization of pMT1 has identified five genes that may be involved in the synthesis of murine toxin (MT) and F1 capsule antigen, both known virulence factors. Expression of both the capsular protein and murine toxin genes has been characterized with respect to environmental cues (e.g., temperature and-calcium) (Du, et al.
Contrib. Microbial. Immunol
. 13:321-324, 1995). F1 capsule synthesis is maximal at 37° C. in the absence of extracellular calcium, conditions similar to those that induce expression of a major
Y. pestis
virulence determinant (Straley
Rev. Infect. Dis
. 10:S323-S326, 1988; Straley
Microbial. Pathoaen
10:87-89, 1991; Straley et al.
Proc. Natl. Acad. Sci. USA
78:1224-1228, 1981). Murine toxin expression is induced at 26° C., conditions similar to those that would be expected to occur in the flea vector. The occurrence of plasmid genes that are induced under widely different conditions suggests regulation of
Y. pestis
virulence determinant expression by at least two networks.
The plasmid pCD1 is found in
Y. pestis
, as well as in certain other pathogenic Yersinia species, including
Y. pseudotuberculosis
and
Y. enterocolitica
. The plasmid encodes a complex virulence property called the low-Ca
2+
response (LCR). The LCR was discovered in
Y. pestis
growing in vitro, where the bacteria respond to the absence of Ca
2+
at 37° C. by the strong expression and secretion of a virulence protein called V antigen, or LcrV. In certain media, expression of LcrV is accompanied by a response termed “restriction,” in which the yersiniae undergo an orderly metabolic shutdown and cease growth. Under LCR-inductive conditions, the transcription, translation, and secretion of a set of virulence proteins called Yops (for
Y
ersinia
o
uter
p
rotein
s
) is maximally induced. The operons encoding these and other similarly regulated operons on the LCR plasmid have been referred to as the LCR stimulon (LCRS). Millimolar concentrations of Ca
2+
permit full growth at 37° C., reduced expression of LcrV and Yops, and essentially no secretion of these proteins. Under ambient temperature conditions outside a mammalian host, the Yops and LcrV proteins are produced at a low, basal level and are not secreted, which suggests that the LCR is designed to function within a mammal. Expression of LCR is apparently modulated by other environmental factors, including Mg
2+
, Cl
−
, Na
+
, glutamate, nucleotides, and anaerobiosity. The molecular basis for these effects has not been determined, but these elements of environmental modulation could be important in adjusting virulence protein expression and secretion in response to the wide range of niches that yersiniae are expected to encounter during an infection.
The pCD1 plasmid also encodes a type III secretion system called Ysc (f

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