As of 2024.3

This report should be submitted within 2 weeks after you return to Japan. Please do not change the formatting（Abroad・Domestic）Internship report form（Student）

2024/07/19(Year/Month/Day)

Name
Laboratory
Year (Grade)
Internship institution
Internship period

KAMBOYI Harvey Kakoma
Division of Infection and Immunity, International Institute for
Zoonosis Control
D4
Hudson Institute of Medical ResearchInternship period:

05/15/2024 -07/07/2024

Departure Date from Sapporo: 05/13/2024, Arrival Date in Sapporo: 07/08/2024)-

Purpose and the reason why you chose this instituteMy internship aimed to acquire training and conduct collaborative research on detailed
molecular analysis of host-pathogenic bacteria interactions. In my future work, I will be
involved in zoonosis research and control. Therefore, it is essential to acquire knowledge
at a molecular level of the interaction of hosts (humans and animals) and pathogens. In
addition, collaborative efforts are necessary to control infectious diseases; thus, theHudson Institute of Medical Research (HIMR) was an ideal place for my internship. The
HIMR is a leading Australian medical research institute recognized internationally fordiscovery science and translational research. The institute strives to improve humanhealth through groundbreaking, collaborative medical research discoveries and thetranslation of these to real-world impact. Further, the HIMR chief executive officer, Prof.
Elizabeth Hartland, who also leads the Innate Immune Responses to Infection researchgroup at CIIID, is an international leader in the fields of microbiology and immunology.
Most notably, she has contributed significantly to understanding underlying host andbacterial molecular mechanisms that determine the outcome of bacterial interactions withhuman cells and disease development. Therefore, combining the research profile of Prof.
E. Hartland and the scope of work being done at the CIIID made me choose HIMR as asuitable site for an internship.

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Result of the activity （about 800 words, provide photos, tables and figures that clearly show theactivities during the period）

1. Orientation and general induction at HIMR

The HIMR is partnered with Monash University and Monash Health and is co-located at

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both organizations at the Monash Health Translation Precinct. Therefore, orientation was
conducted at Monash University and Health (Hospital) (Fig. 1A). I was introduced to thetwelve (12) specialist research groups and their respective laboratories at the Centre forInnate Immunity and Infectious Diseases (CIIID). I was stationed in the Innate Immune
Response to Infection (Hartland Laboratory). After that, I went through the Occupational
Health, Safety and Environment (OHSE) and local area induction. The OHSE assists withorienting new staff and students into the institute, providing familiarity with the local
area, explaining procedures, and providing details of whom to contact for more
information. Induction forms were signed after completing the OHSE and local area
induction (Fig. 1B, C). Further, in view of the collaborative research I was going to be
involved in, I also signed the intellectual property deed poll (Fig. 1D). To access spaces andfacilities at HIMR, I was given access keys to Monash University facilities as voluntary
staff and Monash Health as a research student (Fig. 1E, F). In addition, I was given an
official HIMR email, which I used to access online resources provided by Monash
University and Monash Health.

Figure 1. Orientation and induction activities. A.
Orientation at Monash Health.
B. Completed and signed OHSE induction form,
Local area (Hartland laboratory) induction form(C) and intellectual property deed pool form
(D).

This activity made me appreciate the collaborative network at HIMR and theircontributions towards improving public health through groundbreaking, collaborative
medical research discoveries and the translation of these to real-world impact.

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2. Training on handling and caring for mammalian cell lines for infection

In the Hartland laboratory, their work is aimed at investigating the manipulation of hostcell signaling by effector protein families and understanding their influence on host cellfunction, inflammatory signaling, and the innate immune response. In this way, effector
proteins can be used as tools to understand the innate responses that are important forthe control of pathogens. The research involves infecting various mammalian cell lines
with pathogenic organisms or their isogenic mutants, including Burkholderia, Shigella, E.

coli, Salmonella, Legionella, and Lentivirus. To get the insights and skills needed to
conduct these research activities, first, I had to familiarize myself with and understandthe standard operating procedures (SOPs) for each mammalian cell line and pathogenicorganism. After showing adequate knowledge and competency as judged by mysupervisors, I was allowed to have hands-on experience through experimentation (Fig. 2A).
I was involved in THP1 monocytes resuscitation from liquid nitrogen, growing them to thepoint of splitting for the next passage and seeding them for infection experiments. I
learned how to stain and enumerate THP1 monocytes using a Neubauer hemocytometer,
which is required to split or seed a fixed number of THP1 monocytes. THP1 monocytes canbe chronically infected with Mycoplasma, which interferes with planned infection
experiments. Therefore, resuscitated THP1 monocytes had to be routinely screened for

Mycoplasma. I conducted Mycoplasma testing using the MycoStrip™ - Mycoplasma
detection kit on the THP1 monocytes assigned to me; fortunately, they tested negative (Fig.
2B and 2C).

Figure 2. Some of the laboratory activities. A. Preparation for splitting and seeding THP1 monocytes in a PC
level 2 clean room. B. Conducting a MycoStripTM assay. C. Results of the MycoStripTM assay.

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To seed THP1 monocytes, we added phorbol 12-myristate 13-acetate which halted divisionof cells but induced their differentiation into mature macrophages and increased cell
adherence to the floor of the bottom of the 24-well plate (Fig. 3A). The adherent monolayerof THP1 macrophages were easily studied for morphological changes that resulted from
infection with a pathogenic organism (Fig. 3B). Morphological changes were observed
using the EVOS™ M5000 Cell Imaging System. This imaging system was also used to
track fluorescent-tagged effector proteins produced by recombinant pathogenic organisms
in the host cellular compartment (Fig. 3C). This gave insights into the subcellular level ofinteraction between the pathogen and the host cells. This level of training prepared meadequately for my planned Bacillus cereus infection in my PhD experiments.

Figure 3. Viewing THP1 cells using the EVOS™ M5000 Cell Imaging System. A. Uninfected THP1 cells. B.
Infected THP1 cells. C. Viewing of cells infected with a GFP-tagged effector protein.

3. Conducting pathogen-specific risk assessment and developing SOPs

In fulfillment of the HIMR guidelines on OHSE, potential risks of any laboratory
procedure must be identified and evaluated to determine risk controls. Thereafter, SOPs
must be developed for everyone working with or within the laboratory to follow after priortraining. As part of the induction process, my supervisors ensured I acquainted myself
with relevant SOPs within the Hartland laboratory before I could be involved in any
laboratory work. Therefore, given the SFDA to be conducted at the same site, I was
required to conduct a risk assessment and develop an SOP for B. cereus experiments in a
PC2 laboratory. The CIIID had no prior research on B. cereus, therefore, its risk
assessment had not been done, and SOPs were not in place. Given my long experience
working with Bacillus species, I led the team from among laboratory members to conduct
the risk assessment (Fig. 4A) and develop the SOP (Fig. 4B) for B. cereus. Beyond myinternship and SFDA at CIIID, we identified a key person within the Hartland laboratory
who would oversee all future collaborative research activities on B. cereus. This activity
demonstrated the importance of public health preparedness and collaboration in achievingAs of 2024.3

research goals. I believe this mindset is essential in my future career as a zoonosis control
expert.

Figure 4. Documents developed during my internship. A. B. cereus risk assessment. B. B. cereus SOP for
infection of THP1 macrophages.

4. Other activities

I attended meetings and research seminars hosted by HIMR almost every Thursday from12:00 to 13:00. The presenters included local and international researchers with aninternational perspective on current global advances in research. The seminars gave mean opportunity that significantly enriched my understanding of global health problems
and solutions for effective pathogen control and prevention (Fig. 4A). I also attended theHartland laboratory research (Thursday 10:00 to 12:00) (Fig. 4B) and social meetings(Tuesday 14:00) (Fig. 4D. In research meetings, PhD students, postdocs, and researchscientists presented their scheduled research progress. I was also given an opportunity topresent my internship progress (Fig. 4B). I also participated in a laboratory field trip in
the countryside of central Victoria state (Fig. 4C) and Philip Island.

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Figure 4. Hartland laboratory activities. A. Attending an HIMR seminar with lab members and Prof. E.
Hartland. B. My progress presentation during the lab meeting. C. Field trip to the countryside in Victoria
state. D. Social lab meeting.

What do you think the positive impact of the activity will have on your further
career path?
My career path will involve researching infectious diseases and their control in Zambia
and internationally, dealing with laboratory and field work. I believe the research network
I have been exposed to at HIMR will play a crucial part in actualizing my career plan. The
HIMR values that include innovation and partnerships have led the institute to bring
together 457 scientists from 29 countries, providing me an opportunity to establish
researcher links from Australia, German, Nepal, China, and Ethiopia. In addition, myinternship activities have led to a research partnership and collaboration between HIMR
and my laboratory, the Division of Infection and Immunity, on B. cereus research for the
next five years. This partnership will significantly help me develop and advance myresearch career, working at the international level.

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Report how your activity could link to One Health Approach (If applicable)

If you also conducted OH onsite training (Ally Module 4), please describe some of the examples of
One Health approach you implemented in your activity. Or explain the possibility(ies) how you
could link this activity to One Health approach for your future.

The activity conducted during the internship training significantly aligns with theprinciples of the One Health approach, which emphasizes the need for collaborative
research and networking among professionals from human, animal, and environmental
sectors. For Ally Module 4, I conducted collaborative research on B. cereus withindividuals from the biomedical sciences and human health sectors originating fromdifferent countries, given my background in veterinary medicine. B. cereus is anenvironmental pathogen that causes infection in humans and animals; hence, it is a

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zoonotic pathogen and a One Health problem.

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Advice for your junior fellows-

We are fortunate to receive financial support from WISE and other sources, however,
we have to pay accommodation, visa, and travel insurance fees upfront and wait for
reimbursement later on. Therefore, you need to save enough to cover these fees. If you
plan to conduct an internship and SFDA simultaneously at the same site, theaccommodation fee may be too much to be up front by yourself. In this case, pleaseengage your laboratory to assist you well in advance.

Institution・Official title・NameApproval of supervisor

International Institute for Zoonosis Control, Division of
Infection and Immunity. Professor. Hideaki HIGASHI※1※2

A certification form from the host should also be submitted.
The Career Path Committee will first confirm the content of this report and report will be forwarded to the
Educational Affairs Committee for credits evaluation.

Hokkaido University
WISE Program for“One Health Frontier Graduate School of Excellence”
One Health Ally Course
Student Free Design Activities Report form

北海道大学
One Health フロンティア卓越大学院プログラム
One Health Allyコース
Student Free Design Activities報告書

Student Free Design Activities (One Health on-site Training)
報告書 Report
報告者 [Reporter]
氏名 [Full Name]
学年 [Year]所属 [Affiliation]

KAMBOYI Harvey Kakoma
D4E-mailGraduate School of Infectious Diseases

担当教員 [Instructor]
氏名 [Full Name]

Elizabeth Hartland

署名 [Signature]

所属 [Affiliation]

Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research,
Melbourne, Australia

E-mail活動報告 [Activity Report]※活動内容が判る様な写真や図表を加えて下さい。/ Provide photos, tables and figures that clearly show the activities during the period.

タイトル [Course Title]

Analysis of Bacillus cereus T7SS-dependent host interactions

実施期間 [Periods]

10/06/2024 – 05/07/2024

共同実施者[Other participants]

Nil

言語 [Language]
実施場所 [Location]

EnglishCentre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research,
Melbourne, Australia

申請時計画の実施報告 [Report how you carried out your plan in the application form]
I followed the schedule of activities according to the plan with minor modifications based on the initial results of theplanned collaborative research. Hence, the transcriptome component of the activity could not be completed within theStudent Free-Design Activity (SFDA) period, but our collective efforts will continue in Australia and Japan. This
collaborative research will augment my PhD research project on Bacillus cereus.
Prior preparation
To conduct this collaborative research on B. cereus, research permission was first sought from the Monash University
Institutional Biosafety Committee. The approval certificate was granted (IBC Reference 34934), and permission toimport B. cereus strains from Japan was also given by the Department of Agriculture, Fisheries and Forestry of theAustralian Government (Permit: 0007003103). Further, Hokkaido University approved the transfer (Approval: 2022027) of B. cereus strains transfer to Monash University for research. The strains were successfully shipped from
Hokkaido University to Monash University, Hudson Institute of Medical Research (HIMR). The B. cereus strains were
resuscitated upon arrival, and glycerol stocks were prepared for long-term storage.
Experiments on B. cereus infection of THP1 cells
The Centre for Innate Immunity and Infectious Diseases (CIIID) at HIMR had no prior research on B. cereus.
Therefore, we drafted the SOP for B. cereus infection of THP1 macrophages and revised it as we optimized the北海道大学One Health フロンティア卓越大学院プログラム
One Health Allyコース
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Hokkaido University
WISE Program for“One Health Frontier Graduate School of Excellence”
One Health Ally Course
Student Free Design Activities Report form

conditions for infection experiments. In addition, we conducted the risk assessment for the Occupational Health,
Safety, and Environmental hazards for working with B. cereus in the PC 2 laboratory.
In setting up the conditions for infection, we used the B. cereus wild-type strain and its isogenic type VII secretion
system knockout strain (ΔessC). The overall objective of these experiments was to obtain THP1 macrophage response
from B. cereus infection that can be measured cytometrically and allow for transcriptome analysis. In summary, we
titrated the B. cereus multiplicity of infection (MOI) from 5 to 0.1 following the protocol illustrated in Figure 1.

Figure 1. Schematic representation of the protocol for infecting THP1 macrophages with B. cereus strains.

Unfortunately, almost 90% of THP1 macrophages died within 4 hours post-infection at the lowest possible dose of
MOI = 0.1 that would produce a consistent cellular response (Fig. 2a). We observed that B. cereus was capable ofmultiplying too fast in mammalian cell culture media (Fig. 2b). The quick death rate of THP1 macrophages would
affect downstream analysis, especially transcriptome analysis. Therefore, we changed the approach of infecting the
THP1 macrophages from using actual bacteria to bacterial culture supernatant. We have already established that theT7SS-dependent effectors are secreted into the culture medium.

A

B

Figure 2. B. cereus strains caused rapid death of THP1 macrophages. A. Representative brightfield images of THP1 macrophagesat MOI = 0.1 with and without tetracycline. B. Dead and live THP1 cells are encircled in red and white rings. B. cereus can beseen as numerous rod-shaped features in the entire image.

THP1 macrophages were consequently exposed to different doses of B. cereus WT and ΔessC filtered cultureHokkaido University
WISE Program for“One Health Frontier Graduate School of Excellence”
One Health Ally Course
Student Free Design Activities Report form

北海道大学
One Health フロンティア卓越大学院プログラム
One Health Allyコース
Student Free Design Activities報告書

supernatants and then observed for observable responses microscopically (Fig. 3a).

A

B

Figure 3. B. cereus supernatants caused death of THP1 macrophages in a dose-dependent manner. A. Representative time-lapse
images of THP1 macrophages dosed with 10% and 1% of WT and ΔessC culture supernatant. Affected misshaped cells canbe seen from 1 h post infection at 10% dose B. Viewing cells using the Invitrogen™ EVOS™ M5000 Cell Imaging System.

Further, we assessed cell death using the lactate dehydrogenase assay, which measures lactate dehydrogenase
concentration in the cell culture supernatant released by dying THP1 macrophages (Fig 4). The results showed a dosedependent response of THP1 macrophages to B. cereus WT and ΔessC supernatants extended up to 20 hours of
incubation.

B

A

D

3h post-treatment

E

WT supernatant

6h post-treatment

C

ΔessC supernatantF

Figure 5. Measurement of cell death represented by LDH release. A. Spectrophotometric measurement of samples using the
CLARIOstar PlusTM Microplate Reader. B, C. LDH release in response to LB media or RPMI + 10% FBS culture supernatantsfrom the WT and ΔessC mutant after 6 h. D, E and F. Time course assessment of LDH release after treatment with varying dosesof RPMI + 10% FBS B. cereus culture supernatant.

In summary, preliminary results for B. cereus WT and ΔessC culture supernatants’ effect on THP1 macrophages weresimilar. At this stage, we could not rule out the influence of other toxins not dependent on T7SS that, in the same way,
kill THP1 macrophages, which are present in the two strains we used in our experiments. From the experiments
conducted so far, we have created an initial library of cell culture supernatants that will be used to treat cells forcytometric and transcriptome analysis.

北海道大学One Health フロンティア卓越大学院プログラム
One Health Allyコース
Student Free Design Activities報告書

Hokkaido University
WISE Program for“One Health Frontier Graduate School of Excellence”
One Health Ally Course
Student Free Design Activities Report form

This collaborative research is continuing with activities being implemented in Australia and Japan.

目的達成状況報告 [Report how you achieved your goal/objectives listed in the application form]
The first objective was achieved, while the second was partially completed. We successfully implemented
collaborative research on B. cereus, a zoonotic pathogen. This objective will continue to be implemented till weestablish the role that the T7SS plays in host infection and possibly further. The second objective to establish the rolethat T7SS plays in host infection and the nature of innate immune response mounted could not be achieved within thefour weeks because we had to establish and optimize the infection conditions first as explained above. Fortunately,
this objective will be achieved since the collaborative research is still ongoing.
One Health Approach実践報告 [Report how your activity could link to One Health Approach]Firstly, B. cereus is an environmental pathogen that causes infection in humans and animals; hence, it is a zoonotic
pathogen and a One Health problem. In addition, T7SS among Gram-positive bacteria is necessary for niche adaptation
and ultimately crucial for survival on hospital and equipment surfaces that serve as sources of nosocomial infections
that are challenging to treat because such bacteria, including B. cereus, become resistant to antimicrobials that would
otherwise kill them. Thus, B. cereus links the three sectors of One Health: human, animal, and environmental healthsectors. Secondly, I had the privilege of working with and collaborating with professionals from different backgrounds
on a One Health-related pathogen. My background is in the veterinary sector, and I conduct collaborative research on
B. cereus with individuals from the biomedical sciences and human health sectors and different nationalities. One
Health calls for collaborative research of multiple disciplines, and I am happy that I have implemented that at theinternational level.
備考 [Remarks]※ 報告書を作成後、担当教員に確認をお願いし署名をもらってください。PDFファイルとしてVetlogから提出してください。
提出先：「Student Free Design Activities報告書」※ Please ask your instructor to check this report and get his/her signature. The scanned report is to be submitted through
Vetlog「Student Free Design Activities Report」.