Preface
This book is devoted to methods in molecular biology to study Leptospira, a fascinating
group of bacteria, and their unique properties. Leptospira spp. are spirochetes that are
comprised of pathogenic and saprophytic species. Saprophytes ubiquitously exist in the
environment such as soil and water. On the other hand, pathogens, the causative agents of
leptospirosis, a zoonotic disease with more than 1 million cases per year, colonize the
proximal renal tubules of mammals and some other vertebrates. Pathogenic Leptospira
spp. are excreted in the urine of reservoir hosts and contaminate environments, where
they can survive for weeks. Humans and animals are infected with leptospires by exposure
to water or soil contaminated with the urine of infected animals or direct exposure to the
urine or tissues of infected animals.
The aim of this book is to provide tips for the manipulation of Leptospira spp. and the
investigation of pathogenesis of leptospirosis based on cutting-edge experimental protocols.
This book provides researchers detailed practical procedures and the Methods section
describes comprehensive step-by-step procedures for each protocol. The Notes section is
intended to give additional information and troubleshooting guides.
Leptospira spp. are slow-growing bacteria requiring rich media and several weeks of
incubation for growth, and their isolation from environmental or biological samples is
fastidious and challenging. Chapter 1 provides the procedure for cultivating and isolating
leptospires from both clinical and environmental samples. The availability of whole genome
sequences, as a result of improved methodology and reduced cost, has resulted in major
improvements in the understanding of the spirochete biology, and whole genome sequencing (WGS) has emerged as an ultimate powerful tool for bacterial strain classification and
epidemiological typing (Chapter 2). In the medical microbiology laboratory, MatrixAssisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry is also an
emerging tool for the identification of bacterial species, and this method can be applied
for the identification of Leptospira species (Chapter 3).
Leptospira have unique morphological features. They exhibit thin, long, and helixshaped cells. They also possess surface-exposed lipopolysaccharide (LPS) and periplasmic
flagella (or endoflagella). Protocols for cell enumeration by flow cytometry (Chapter 4),
RNA preparation (Chapter 5), and purification of LPS (Chapter 6) as well as a protocol for
phage purification (Chapter 7) are included in this volume.
In comparison to other bacteria, our knowledge of the molecular basis of the pathogenesis of leptospirosis is limited. This is largely due to the fact that genetic manipulation of
Leptospira was not possible or not efficient. Today major advances achieved in genetics and
in all steps of functional studies in Leptospira spp. provide us with an opportunity to apply
state-of-the-art approaches for the identification and characterization of virulence factors.
Random transposon mutagenesis can be applied to both saprophytes and pathogens
(Chapter 8). Transposon sequencing (Tn-seq) that combines transposon insertional mutagenesis with high-throughput sequencing of the transposon insertion sites is able to identify
genes contributing to virulence in pathogens (Chapter 9). More recently, specific gene
silencing using RNA-guided catalytically inactive Cas9 (dCas9) has been successfully used
in L. biflexa (Chapter 10).
v
This book is devoted to methods in molecular biology to study Leptospira, a fascinating
group of bacteria, and their unique properties. Leptospira spp. are spirochetes that are
comprised of pathogenic and saprophytic species. Saprophytes ubiquitously exist in the
environment such as soil and water. On the other hand, pathogens, the causative agents of
leptospirosis, a zoonotic disease with more than 1 million cases per year, colonize the
proximal renal tubules of mammals and some other vertebrates. Pathogenic Leptospira
spp. are excreted in the urine of reservoir hosts and contaminate environments, where
they can survive for weeks. Humans and animals are infected with leptospires by exposure
to water or soil contaminated with the urine of infected animals or direct exposure to the
urine or tissues of infected animals.
The aim of this book is to provide tips for the manipulation of Leptospira spp. and the
investigation of pathogenesis of leptospirosis based on cutting-edge experimental protocols.
This book provides researchers detailed practical procedures and the Methods section
describes comprehensive step-by-step procedures for each protocol. The Notes section is
intended to give additional information and troubleshooting guides.
Leptospira spp. are slow-growing bacteria requiring rich media and several weeks of
incubation for growth, and their isolation from environmental or biological samples is
fastidious and challenging. Chapter 1 provides the procedure for cultivating and isolating
leptospires from both clinical and environmental samples. The availability of whole genome
sequences, as a result of improved methodology and reduced cost, has resulted in major
improvements in the understanding of the spirochete biology, and whole genome sequencing (WGS) has emerged as an ultimate powerful tool for bacterial strain classification and
epidemiological typing (Chapter 2). In the medical microbiology laboratory, MatrixAssisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry is also an
emerging tool for the identification of bacterial species, and this method can be applied
for the identification of Leptospira species (Chapter 3).
Leptospira have unique morphological features. They exhibit thin, long, and helixshaped cells. They also possess surface-exposed lipopolysaccharide (LPS) and periplasmic
flagella (or endoflagella). Protocols for cell enumeration by flow cytometry (Chapter 4),
RNA preparation (Chapter 5), and purification of LPS (Chapter 6) as well as a protocol for
phage purification (Chapter 7) are included in this volume.
In comparison to other bacteria, our knowledge of the molecular basis of the pathogenesis of leptospirosis is limited. This is largely due to the fact that genetic manipulation of
Leptospira was not possible or not efficient. Today major advances achieved in genetics and
in all steps of functional studies in Leptospira spp. provide us with an opportunity to apply
state-of-the-art approaches for the identification and characterization of virulence factors.
Random transposon mutagenesis can be applied to both saprophytes and pathogens
(Chapter 8). Transposon sequencing (Tn-seq) that combines transposon insertional mutagenesis with high-throughput sequencing of the transposon insertion sites is able to identify
genes contributing to virulence in pathogens (Chapter 9). More recently, specific gene
silencing using RNA-guided catalytically inactive Cas9 (dCas9) has been successfully used
in L. biflexa (Chapter 10).
v