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. 2005 Jun;73(6):3251-60.
doi: 10.1128/IAI.73.6.3251-3260.2005.

Changes in lipopolysaccharide O antigen distinguish acute versus chronic Leptospira interrogans infections

Affiliations

Changes in lipopolysaccharide O antigen distinguish acute versus chronic Leptospira interrogans infections

Jarlath E Nally et al. Infect Immun. 2005 Jun.

Abstract

Leptospirosis is the most geographically widespread zoonotic disease in the world. A severe pulmonary form of leptospirosis (SPFL) is being recognized with increased frequency. We have reported that human SPFL isolates of Leptospira cause acute lethal infection with prominent pulmonary hemorrhage in guinea pigs. We have found that the same SPFL strains cause asymptomatic infection and chronic renal shedding in rats, where infection is restricted to the renal tubules. To address the antigenic composition of host tissue-derived Leptospira (HTL), motile leptospires were purified from guinea pig liver by centrifugation on Percoll density gradients and compared to Percoll-purified in vitro-cultivated Leptospira (IVCL). The lipopolysaccharide O antigen (Oag) content of guinea pig liver-derived HTL was markedly reduced compared to that of IVCL, as demonstrated both by immunoblotting with a monoclonal antibody that was serovar specific for Oag and by periodate-silver staining. Confocal microscopy of HTL in guinea pig liver and kidney with the Oag-specific monoclonal antibody provided further evidence that diminution of the Oag content occurred in situ during lethal infection. In contrast, the Oag content of HTL in chronically infected rat renal tubules was indistinguishable from that of IVCL. These findings suggest that there may be regulation of Oag synthesis by Leptospira specific to the animal host infected. The hypothesis that the Oag content is related to whether lethal infection or chronic renal tubular colonization occurs remains to be tested.

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Figures

FIG. 1.
FIG. 1.
Features of acute guinea pig and chronic rat infections with SPFL strain RJ16441. Acutely infected guinea pigs have large numbers of leptospires disseminated in the liver (original magnification, ×ばつ400) (A) and the kidney (original magnification, ×ばつ400) (B), as demonstrated by immunohistochemistry with antiserum specific for OMV of Leptospira. Few inflammatory cells are present. In contrast, immunohistochemistry showed that in chronically infected rats at day 21, the presence of leptospires was restricted to renal tubules (original magnification, ×ばつ100) (C). (D) Hematoxylin and eosin staining of infected rat kidney revealed the absence of inflammatory cells (original magnification, ×ばつ200). (E) Uninfected rat kidney showed no binding of OMV antiserum.
FIG. 2.
FIG. 2.
Antigenic composition of guinea pig liver-derived HTL and demonstration that the content of a proteinase K-resistant 22-kDa antigen is diminished. Immunoblots of equal numbers of whole IVCL cells purified over Percoll (lane 1), whole guinea pig liver-derived HTL cells purified over Percoll (lane 2), Percoll-purified IVCL cells treated with proteinase K (lane 3), and Percoll-purified HTL cells treated with proteinase K (lane 4) were probed with either antiserum specific for OMV of Leptospira (A) or serum from chronically infected rats (CRS) (B). In addition, Percoll-purified IVCL or HTL was immunoblotted with antiserum specific for flagella (C) or OmpL1 (33) (D) to verify that similar amounts of Leptospira cells were loaded in each lane. The integrated density values (IDV) demonstrate that the diminished content of the 22-kDa proteinase K-resistant antigen was not due to smaller amounts of HTL being compared to IVCL. The positions of molecular mass markers (in kilodaltons) are indicated to the left of each blot. An star indicates the position of the 22-kDa antigen expressed in smaller amounts in HTL than in IVCL.
FIG. 3.
FIG. 3.
Comparison of enterobacterial and leptospiral LPS and identification of serovar Copenhageni Oag. (A) Periodate-silver staining of Salmonella rough LPS (2.5 μg) (lane 1), Salmonella smooth LPS (7.5 μg) (lane 2), L. interrogans serovar Copenhageni strain RJ16441 LPS from approximately 7.5 ×ばつ 107 cells (lane 3), L. interrogans serovar Copenhageni strain RJ15958 LPS from approximately 7.5 ×ばつ 107 cells (lane 4), L. kirschneri LPS from approximately 108 cells (lane 5), and L. biflexa LPS from approximately 7.5 ×ばつ 107 cells (lane 6). The positions of molecular mass markers (in kilodaltons) are indicated on the left. (B) Immunoblot of the same samples as in panel A probed with monoclonal antibody F70C24 specific for LPS of serovar Copenhageni.
FIG. 4.
FIG. 4.
Reduced Oag content in guinea pig liver-derived HTL compared to IVCL. Parallel immunoblots of proteinase K-treated Percoll-purified IVCL (lane 1) and HTL (lane 2) were probed with monoclonal antibody F70C24 (A) or CRS (B). In both cases the diminution in Oag content of HTL is apparent. (C) Periodate-silver staining of whole IVCL (lane 1) and HTL (lane 2), proteinase K-treated IVCL (lane 3), and proteinase K-treated HTL (lane 4) shows that no 22-kDa Oag band is seen in proteinase K-treated HTL.
FIG. 5.
FIG. 5.
Oag content of Leptospira in situ in rat renal tubules and in guinea pig livers and kidneys: confocal microscopy of IVCL and HTL. Double-immunofluorescence labeling of IVCL embedded in agar (A, B, and C), HTL in guinea pig liver (D, E, and F), HTL in guinea pig kidney (G, H, and I), and HTL in chronically infected rat kidney (J, K, and L) was performed with anti-OMV, which was detected with an Alexa fluor 488 conjugate (A, D, G, and J), or with monoclonal antibody F70C24, which was detected with a Cy-3 conjugate (B, E, H, and K). Detection of both reagents in the same panel for each sample is also shown (C, F, I, and L). Control images of normal tissues and infected tissue stained without primary antibody are not shown. The length of each side of each panel corresponds to 158.75 μm.
FIG. 5.
FIG. 5.
Oag content of Leptospira in situ in rat renal tubules and in guinea pig livers and kidneys: confocal microscopy of IVCL and HTL. Double-immunofluorescence labeling of IVCL embedded in agar (A, B, and C), HTL in guinea pig liver (D, E, and F), HTL in guinea pig kidney (G, H, and I), and HTL in chronically infected rat kidney (J, K, and L) was performed with anti-OMV, which was detected with an Alexa fluor 488 conjugate (A, D, G, and J), or with monoclonal antibody F70C24, which was detected with a Cy-3 conjugate (B, E, H, and K). Detection of both reagents in the same panel for each sample is also shown (C, F, I, and L). Control images of normal tissues and infected tissue stained without primary antibody are not shown. The length of each side of each panel corresponds to 158.75 μm.

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