days from all sites except the kidney, where they colonize the
proximal renal tubules [3–6]; within this immunoprivileged
niche, leptospires are continuously bathed in nutrient-rich, glomerular ultrafiltrate, which closely resembles interstitial fluid [4, 5,
7]. Importantly, even at high inoculum (e.g., 10
7 organisms), rats
are asymptomatic and, following renal colonization, continue to
shed leptospires in urine for weeks [3–6]. In contrast, hamsters and
guinea pigs, the species most commonly used to study acute leptospirosis [2, 3], are exquisitely sensitive to infection, succumbing to
disease with inocula 10 organisms [8]. These observations imply
that outcomes following infection are determined largely by the
nature and intensity of the host inflammatory response [9–
12]. These data argue that pathogenic Leptospira spp. employ
similar, if not identical, genetic programs to establish themselves
within asymptomatic (i.e., reservoir) and symptomatic (i.e., incidental) hosts despite the markedly divergent immunopathological
events that follow infection. While little is known about how Leptospira spp. persist within mammals, numerous studies suggest that
leptospires alter their transcriptomic and proteomic profiles in
response to environmental signals encountered during infection
[13–24] Although sufficient for growth in vitro, artificial medium
almost certainly does not replicate the full range of environment
signals encountered by leptospires either outside or inside the host.
Analysis of host-adapted leptospires from naturally or experimentally infected hosts is limited by difficulties associated with
recovering intact organisms free from host tissues or other contaminants. To circumvent these issues, we developed a facile model
for generating large numbers of host-adapted L. interrogans within
rat peritoneal cavities [13]. Leptospires diluted to low density
(3000–10,000 per ml) within EMJH medium are placed within
dialysis membrane tubing, tied off at both ends to form a chamber.
The dialysis membrane chamber (DMC) is implanted in the peritoneal cavity of a rat and allowed to incubate for ~7–10 days, during
which time leptospires reach densities (~10
8 per ml) comparable to
those observed with in vitro cultures. The molecular weight cutoff
of the dialysis tubing (8000 Da) allows for rapid exchange of
nutrients and other small molecules within host interstitial fluid
but protects the bacterium from the host immune system. Using
this model, we routinely recover >10
9 viable host-adapted leptospires per DMC.
Because of the large numbers of host-adapted leptospires
recovered, the DMC model provides a facile platform for
genome-wide comparative transcriptomic and proteomic analyses.
Using RNAseq, we identified >160 genes differentially expressed
by L. interrogans serovar (sv.). Copenhageni strain (st.) Fiocruz
L1-130 leptospires in DMCs were compared to their in vitro counterparts [13]. We subsequently reported that the proteome of
DMC-cultivated
leptospires
undergoes
posttranslational
230
Andre Alex Grassmann and Melissa J. Caimano
proximal renal tubules [3–6]; within this immunoprivileged
niche, leptospires are continuously bathed in nutrient-rich, glomerular ultrafiltrate, which closely resembles interstitial fluid [4, 5,
7]. Importantly, even at high inoculum (e.g., 10
7 organisms), rats
are asymptomatic and, following renal colonization, continue to
shed leptospires in urine for weeks [3–6]. In contrast, hamsters and
guinea pigs, the species most commonly used to study acute leptospirosis [2, 3], are exquisitely sensitive to infection, succumbing to
disease with inocula 10 organisms [8]. These observations imply
that outcomes following infection are determined largely by the
nature and intensity of the host inflammatory response [9–
12]. These data argue that pathogenic Leptospira spp. employ
similar, if not identical, genetic programs to establish themselves
within asymptomatic (i.e., reservoir) and symptomatic (i.e., incidental) hosts despite the markedly divergent immunopathological
events that follow infection. While little is known about how Leptospira spp. persist within mammals, numerous studies suggest that
leptospires alter their transcriptomic and proteomic profiles in
response to environmental signals encountered during infection
[13–24] Although sufficient for growth in vitro, artificial medium
almost certainly does not replicate the full range of environment
signals encountered by leptospires either outside or inside the host.
Analysis of host-adapted leptospires from naturally or experimentally infected hosts is limited by difficulties associated with
recovering intact organisms free from host tissues or other contaminants. To circumvent these issues, we developed a facile model
for generating large numbers of host-adapted L. interrogans within
rat peritoneal cavities [13]. Leptospires diluted to low density
(3000–10,000 per ml) within EMJH medium are placed within
dialysis membrane tubing, tied off at both ends to form a chamber.
The dialysis membrane chamber (DMC) is implanted in the peritoneal cavity of a rat and allowed to incubate for ~7–10 days, during
which time leptospires reach densities (~10
8 per ml) comparable to
those observed with in vitro cultures. The molecular weight cutoff
of the dialysis tubing (8000 Da) allows for rapid exchange of
nutrients and other small molecules within host interstitial fluid
but protects the bacterium from the host immune system. Using
this model, we routinely recover >10
9 viable host-adapted leptospires per DMC.
Because of the large numbers of host-adapted leptospires
recovered, the DMC model provides a facile platform for
genome-wide comparative transcriptomic and proteomic analyses.
Using RNAseq, we identified >160 genes differentially expressed
by L. interrogans serovar (sv.). Copenhageni strain (st.) Fiocruz
L1-130 leptospires in DMCs were compared to their in vitro counterparts [13]. We subsequently reported that the proteome of
DMC-cultivated
leptospires
undergoes
posttranslational
230
Andre Alex Grassmann and Melissa J. Caimano